ENZYMATIC CONVERSION OF PRETREATED BIOMASS INTO FERMENTABLE
SUGARS FOR BIOREFINERY OPERATION
By
Dahai Gao

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Chemical Engineering
2011

ABSTRACT
ENZYMATIC CONVERSION OF PRETREATED BIOMASS INTO FERMENTABLE
SUGARS FOR BIOREFINERY OPERATION
By
Dahai Gao
Depleting petroleum reserves and potential climate change caused by fossil fuel consumption
have attracted significant attention towards the use of alternative renewable resources for
production of fuels and chemicals. Lignocellulosic biomass provides a plentiful resource for the
sustainable production of biofuels and biochemicals and could serve as an important contributor
to the world energy portfolio in the near future. Successful biological conversion of
lignocellulosic biomass requires an efficient and economical pretreatment method, high
glucose/xylose yields during enzymatic hydrolysis and fermentation of both hexose and pentose
to ethanol. High enzyme loading is a major economic bottleneck for the commercial processing
of pretreated lignocellulosic biomass to produce fermentable sugars.
Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant
reduction in enzyme loading without sacrificing hydrolysis yield. Core glycosyl hydrolases were
isolated and purified from various sources to help rationally optimize an enzyme cocktail to
digest ammonia fiber expansion (AFEX) treated corn stover. The four core cellulases were
endoglucanase I (EG I), cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and βGlucosidase (βG). The two core hemicellulases were an endoxylanase (EX) and a β-xylosidase
(βX). A diverse set of accessory hemicellulases from bacterial sources was found necessary to
enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High
glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme

loading (~20 mg protein/g glucan) using an in-house developed enzyme cocktail and this
cocktail was compared to commercial enzyme.
Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving
a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and
degradation products formed during pretreatment impede accurate quantification of individual
glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput Fast Protein Liquid
Chromatography (HT-FPLC) based method has been developed to quantify CBH I, CBH II and
EG I present in hydrolyzates of untreated, AFEX, and dilute-acid pretreated corn stover. This
method can accurately quantify individual enzymes present in complex binary and ternary
protein mixtures without interference from plant cell wall derived components.
The binding characteristics of CBH I, CBH II and EG I during 48 hours hydrolysis were studied
on different cellulose allomorphs: microcrystalline cellulose Avicel (cellulose Iβ), liquid
ammonia treated cellulose (cellulose III), sodium hydroxide treated cellulose (cellulose II) and
phosphoric acid swollen amorphous cellulose (AC). The digestibility ranking is AC>cellulose
III>cellulose II>cellulose I. However, AC has the highest initial enzyme binding capacity while
cellulose III had the lowest. CBH II is less stable during hydrolysis. Time course binding studies
were also performed for pretreated biomass. Ammonia Fiber Expansion (AFEX) treated corn
stover (CS), dilute acid (ACID) treated CS and ionic liquid (IL) pretreated CS were compared.
The results indicate that presence of lignin is responsible for significant unproductive cellulase
binding. These results are critical for improving our understanding of enzyme synergism,
productive/unproductive enzyme binding and the role of pretreatment on enzyme accessibility to
lignocellulosic plant cell walls. The results also assist in engineering novel low unproductive
binding enzyme systems and developing economic enzyme recycle options.

This work is dedicated to my wife Haiyan Cen and my son Nicholas Jiatong Gao

iv

ACKNOWLEDGMENTS

I’d like to thank my dear advisor, Dr. Bruce E. Dale, who guided me and inspired me all through
my exciting Ph.D study. He also taught me how to efficiently work within a group and contribute
my skill to fulfill my value. In addition, as a role model, he showed me how much achievement
an individual can reach by holding his dream and diligent hard work.
I am very happy to be able to work with so many talent colleagues in my group. Special thanks
to Shishir Chundawat, who always helped me to discover the value in my work, Venkatesh
Balan, who help me get the indispensible resources for my work. All other group members
Rebecca Garlock, Mingjie Jing, Leonardo da Costa Sousa, Pragnya Lavanya Eranki, Bryan Bals,
Elizabeth Sendich and Ming Lau have given me a lot of technical and life support. Many thanks
to our hard working technicians, Nirmal Uppugundla, Christa Gunawan, James Humpula and
Margie Magyar, their contributions helped make my research move quickly. In addition our
visitiors from the entire world, Xiurong Yu, Bensheng Liu, Tongjun Liu, Albert Cheh and
Chandraraj Krishnan also give me invaluable support and suggestions. Thanks to MBI for access
their facilities.
Last, I’d like to thank my family. My wife, Haiyan Cen, who is also my soul mate and study
partner, gave me suggestions and support. And my parents, although far away from East Lansing,
teach me how to work with diligent attitude and how to face and conquer difficulties. Their love
and support is invaluable and priceless.

v

TABLE OF CONTENTS
LIST OF TABLES ....................................................................................................................... X
LIST OF FIGURES .................................................................................................................. XII
CHAPTER 1 INTRODUCTION ............................................................................................... 1
1.1
Background ...................................................................................................................... 1
1.2
Objectives ......................................................................................................................... 2
CHAPTER 2 LITERATURE REVIEW ................................................................................... 3
2.1
Lignocellulosic biomass ................................................................................................... 3
2.1.1
Cellulose ................................................................................................................... 4
2.1.2

Hemicellulose ............................................................................................................ 6

2.1.3

Lignin ........................................................................................................................ 6

2.2
Pretreatment of biomass ................................................................................................... 7
2.2.1
Dilute acid pretreatment ........................................................................................... 8
2.2.2

Liquid hot water pretreatment .................................................................................. 8

2.2.3

Lime pretreatment ..................................................................................................... 9

2.2.4

Ammonia recycle percolation ................................................................................... 9

2.2.5

Ammonia Fiber Expansion ..................................................................................... 10

2.2.6

Ethanol Organosolv ................................................................................................ 11

2.3
Enzyme systems ............................................................................................................. 11
2.3.1
Cellulase ................................................................................................................. 11
2.3.2

Hemicellulase .......................................................................................................... 12

2.3.3

Other proteins ......................................................................................................... 13

2.4
Enzyme synergism ......................................................................................................... 13
2.5
Major challenges of enzymatic hydrolysis ..................................................................... 15
2.5.1
High cost of commercial enzymes ........................................................................... 15
2.5.2

Low activity of enzymes .......................................................................................... 16

2.5.3

Hydrolysis rate decreasing and lower sugar yield at high solid loading ............... 16

2.6
Current progress on enzymatic hydrolysis ..................................................................... 18
2.6.1
Increase substrates digestibility by pretreatment ................................................... 18
2.6.2

Hydrolysis of pretreated biomass using crude enzyme mixtures ............................ 18

2.6.3

Hydrolysis studies by using purified enzymes ......................................................... 23

2.6.4

Enzyme binding characteristics .............................................................................. 25

2.6.5

Others ...................................................................................................................... 28

CHAPTER 3 GLYCOSYL HYDROLASES EXPRESSION AND PURIFICATION ....... 28
vi

3.1
Introduction .................................................................................................................... 28
3.2
Enzyme production ........................................................................................................ 29
3.2.1
Organism................................................................................................................. 29
3.2.2

Culture media.......................................................................................................... 30

3.2.3

Preparation of seed culture .................................................................................... 31

3.2.4

Expression of recombinant enzymes in shake flasks ............................................... 31

3.2.5

Fermentation ........................................................................................................... 31

3.2.6

Production of recombinant enzymes from P. pastoris ............................................ 32

3.2.7

Purification of recombinant enzymes...................................................................... 34

3.3
Protein purification ......................................................................................................... 35
3.3.1
SDS-PAGE and protein purity quantification......................................................... 39
3.3.2

Purification of glycosyl hydrolases from crude enzyme blends .............................. 39

CHAPTER 4 MIXTURE OPTIMIZATION OF SIX CORE GLYCOSYL HYDROLASES
FOR MAXIMIZING SACCHARIFICATION AFEX PRETREATED CORN STOVER.. 44
4.1
Introduction .................................................................................................................... 44
4.2
Materials and Methods ................................................................................................... 45
4.2.1
AFEX pretreatment ................................................................................................. 45
4.2.2

Crude enzyme mixtures ........................................................................................... 45

4.2.3

Heterologous enzyme expression ............................................................................ 46

4.2.4

Protein purification................................................................................................. 46

4.2.5

Proteomics analysis ................................................................................................ 46

4.2.6

Enzyme activity assays ............................................................................................ 47

4.2.7

Enzymatic hydrolysis of AFEX treated corn stover ................................................ 48

4.2.8

Glucose and xylose assays ...................................................................................... 49

4.3
Results and Discussion ................................................................................................... 50
4.3.1
Activity of purified enzymes .................................................................................... 50
4.3.2

Hydrolysis of AFEX treated corn stover by purified enzymes ................................ 53

4.3.3

Optimal core enzyme mixture for hydrolyzing AFEX treated corn stover.............. 61

4.4

Conclusion...................................................................................................................... 71

CHAPTER 5 STRATEGY FOR IDENTIFICATION OF NOVEL FUNGAL AND
BACTERIAL GLYCOSYL HYDROLASE HYBRID MIXTURES ..................................... 72
5.1
Introduction .................................................................................................................... 72
5.2
Materials and Methods ................................................................................................... 73
5.2.1
AFEX pretreatment ................................................................................................. 73
5.2.2

Discovery and cloning of LX1, LX2, LβX and LβG................................................ 73

5.2.3

Discovery and cloning of LC1 and LC2 ................................................................. 73
vii

5.2.4

Enzyme expression and purification ....................................................................... 76

5.2.5

Strategy for enzyme screening on realistic lignocellulosic substrates ................... 76

5.2.6

Enzyme activity assays ............................................................................................ 77

5.2.7

Enzymatic hydrolysis of pretreated biomass........................................................... 77

5.2.8

Glucose and xylose assays ...................................................................................... 77

5.3
Results and discussions .................................................................................................. 78
5.3.1
Specific activities of bacterial and fungal enzymes ................................................ 78
5.3.2

Doping and swapping bacterial/fungal cellulases .................................................. 80

5.3.3

Doping of bacterial hemicellulases to fungal benchmark mixture ......................... 83

5.3.4

Enzyme mixture optimization .................................................................................. 86

5.3.5

Relationship between glucan and xylan conversions.............................................. 94

5.3.6

Discussion ............................................................................................................. 100

5.4

Conclusion.................................................................................................................... 101

CHAPTER 6 HEMICELLULASES AND AUXILIARY ENZYMES FOR IMPROVED
CONVERSION OF LIGNOCELLULOSIC BIOMASS TO MONOSACCHARIDES ..... 103
6.1
Introduction .................................................................................................................. 103
6.2
Materials and Methods ................................................................................................. 104
6.2.1
AFEX pretreatment ............................................................................................... 104
6.2.2

Discovery and cloning of LX1, LX2, LβX and LβG.............................................. 104

6.2.3

Discovery and cloning of L-arabinofuranosidase (LArb) .................................... 104

6.2.4

Discovery and cloning of LX3, LX4, LX5, LX6 and LαGl .................................... 104

6.2.5

Enzyme expression and purification ..................................................................... 108

6.2.6

Enzymatic hydrolysis of pretreated biomass......................................................... 108

6.2.7

Glucose and xylose assays .................................................................................... 108

6.3
Results and discussion.................................................................................................. 108
6.3.1
Supplementing bacterial hemicellulases along with fungal cellulases ................. 108
6.3.2

Screening endoxylanases ...................................................................................... 113

6.3.3

Optimum cellulase to xylanase ratio..................................................................... 114

6.3.4

Synergism between endoxylanases (LX3 and LX4) .............................................. 116

6.3.5

Minimizing loading of accessory enzymes (LβX, LαGl and LArb) ....................... 117

6.3.6
Comparison of monosaccharides yield for optimized and crude commercial enzyme
preparations......................................................................................................................... 118
6.4

Conclusion.................................................................................................................... 122

CHAPTER 7 INTERACTION OF TRICHODERMA REESEI CELLULASES WITH
UNTREATED, AFEX, AND DILUTE-ACID PRETREATED BIOMASS ........................ 124
7.1
Introduction .................................................................................................................. 124
viii

7.2
Materials and Methods ................................................................................................. 125
7.2.1
AFEX pretreatment ............................................................................................... 125
7.2.2

Dilute acid pretreated corn stover ........................................................................ 126

7.2.3

Enzyme purification .............................................................................................. 126

7.2.4

Cellulase Binding and Quantitation ..................................................................... 126

7.2.5

Langmuir Model Fitting........................................................................................ 131

7.3
Results and Discussion ................................................................................................. 131
7.3.1
FPLC Quantification ............................................................................................ 131
7.3.2

Binding isotherms for single, binary and ternary cellulase mixtures ................... 134

7.3.3

Modeling Enzyme Binding Properties .................................................................. 146

7.3.4

Impact of AFEX pretreatment on cooperative cellulase binding.......................... 148

7.4

Conclusions .................................................................................................................. 149

CHAPTER 8 CELLULOSE CRYSTALLINITY AND LIGNIN: THEIR IMPACT ON
EFFICIENT CELLULASE ACTION .................................................................................... 151
8.1
Introduction .................................................................................................................. 151
8.2
Materials and Methods ................................................................................................. 153
8.2.1
Cellulosic substrates ............................................................................................. 153
8.2.2

AFEX pretreated corn stover ................................................................................ 153

8.2.3

Dilute acid pretreated corn stover ........................................................................ 154

8.2.4

Ionic liquid pretreated corn stover ....................................................................... 154

8.2.5

Cellulase Purification ........................................................................................... 154

8.2.6

Enzymatic Hydrolysis............................................................................................ 154

8.2.7

Sugar analysis ....................................................................................................... 155

8.2.8

Quantitation of unbound CBH I, CBH II and EG I .............................................. 155

8.2.9

Thermostability of CBH I, CBH II and EG I ........................................................ 155

8.3
Results and Discussion ................................................................................................. 155
8.3.1
Cellulase hydrolytic activity on different cellulosic substrates ............................ 155
8.3.2

Cellulase binding to different cellulosic substrates during course of hydrolysis . 156

8.3.3

Trichoderma cellulases thermostability................................................................ 161

8.3.4

Cellulase binding and hydrolytic activity on pretreated lignocellulosic biomass 162

8.4

Conclusion.................................................................................................................... 171

CONCLUSION AND SUGGESTIONS FOR FUTURE RESEARCH ................................ 172
REFERENCES .......................................................................................................................... 175

ix

LIST OF TABLES

Table 2.1 Major enzymes and their functions on cell wall polymers. .......................................... 13
Table 2.2 Comparison of ethanol produced from corn kernel and cellulosic biomass ................. 15
Table 2.3 Specific activity for different enzymes compared to commercial cellulases................ 16
Table 2.4 Proteomics data for some commercial enzymes. The abundance of each protein is
reflected by the peptide count number detected during analysis. ................................................. 20
Table 2.6 Currently available cellulase adsorption measurement methodologies ........................ 27
Table 3.1 Chromatographic steps employed during FPLC based purification of various glycosyl
hydrolases from crude enzyme mixtures. ..................................................................................... 37
Table 4.1 Identification of major peptides from six purified glycosyl hydrolases by LC-MS/MS.
....................................................................................................................................................... 51
Table 4.2 Purified enzymes activity assay on chromogenic p-nitrophenyl (pNP) glycosidic
substrates, Avicel, CMC, oat spelt xylan, cellobiose and xylobiose. ........................................... 52
Table 4.3 24 hours hydrolysis yields of AFEX treated corn stover by various enzyme mixtures at
three different total protein loadings. βG was loaded at 10% (mass loading) of all other proteins.
An equivalent amount of Spezyme (equivalent mass basis 33, 16.5 and 8.25 mg/g glucan,
respectively; with no βG supplementation) was included. ........................................................... 58
Table 4.4 Statistical mixture model regression analysis for glucose hydrolysis yields at three total
protein loadings for saccharifying AFEX treated corn stover. ..................................................... 61
Table 4.5 24 hours glucose yield for AFEX treated corn stover saccharified at optimum enzyme
loading. The model generated optimum mixture hydrolysis predictions were tested at varying
total enzyme loadings. .................................................................................................................. 69
Table 5.1 Amino acid sequences and glycosyl hydrolase families for all six bacterial enzymes . 74
Table 5.2 Activity assay data for bacterial and fungal enzymes................................................... 78
Table 5.3 Mixtures of bacterial and fungal enzymes tested on AFEX treated corn stover. The
experimental results for these enzyme combinations are shown in Figure 5.3. ........................... 82
Table 5.4 24 hours glucan and xylan hydrolysis yields of AFEX treated corn stover by various
enzyme mixtures at three different total protein loadings. βG was loaded at 10 (mass loading) of
all other proteins. .......................................................................................................................... 87

x

Table 5.5 Linear regression of xylan vs. glucan conversion at three different total enzyme
loadings. ........................................................................................................................................ 94
Table 6.1 Amino acid sequences and glycosyl hydrolase families for all six bacterial enzymes.
..................................................................................................................................................... 105
Table 7.1 Cooperative and competitive binding among various binary cellulase mixtures for
AFEX pretreated (AFEX-CS), dilute-acid pretreated (ACID-CS) and untreated (UN-CS) corn
stover. Binding types (cooperative versus competitive) are generalized in the table and the
respective enzyme loading ranges within which that particular binding behavior is seen are
shown in brackets. ....................................................................................................................... 144
Table 7.2 Langmuir type (single-site model) binding isotherm parameters for individual cellulase
enzymes binding to untreated (UN-CS), ammonia fiber expansion (AFEX-CS) and dilute acid
treated (ACID-CS) corn stover for various single, binary and ternary enzyme mixtures. ......... 145
Table 8.1 Composition and theoretical concentration of cellulose allomorphs (cellulose I, II, III
and amorphous cellulose) and pretreated corn stover. ................................................................ 164

xi

LIST OF FIGURES

Figure 2.1 Scale model of the polysaccharides in an Arabidopsis leaf cell. Between microfibrils,
the hemicelluloses cross-links (shown in orange) are extended[23]. ............................................. 3
Figure 2.2 Cellulose structures with cellobiose unit linked through β-1,4-glycosic bond.[26] ...... 5
Figure 2.3 Lignin’s three components.(1) p-coumaryl alcohol, (2) coniferyl alcohol and (3)
sinapyl alcohol ................................................................................................................................ 6
Figure 2.4 (A) SDS-PAGE of some commercial enzymes. C: Spezyme CP; G: Novozyme 188; X:
Multifect Xylanase; P: Multifect Pectinase; E: Depol 670L ........................................................ 19
Figure 3.1 SDS-PAGE of expressed recombinant enzymes by P. pastoris during the course of
fermentation. Each lane refers to culture supernatants of different fermentation times in hours.
Each well was loaded with 10 µl of respective supernatant. Where; A - Engoglucanase, B - βGlucosidase, C - β-Xylosidase. Arrows indicate respective enzymes of interest. ........................ 34
Figure 3.2 Separation of protein fractions (I-IV) from commercial enzyme blend (Spezyme CP)
by anion exchange chromatography (Step 1.0) with respect to elution buffer gradient (% B). ... 40
Figure 3.3 Separation of protein fractions (I-IV) from commercial enzyme blend (Novozyme 188)
by anion exchange chromatography (Step 5.1) with respect to elution buffer gradient (% B). ... 41
Figure 3.4 Separation of endo-xylanase from Multifect Xylanase (Step 6.1). Elution profile of
protein UV adsorption (280 nm) with respect to elution buffer gradient. .................................... 42
Figure 3.5 SDS-PAGE of purified EG I (lane 1), CBH II (lane 2), CBH I (lane 3), β-G (lane 4),
EX (lane 5), β-X (lane 6), REG (lane 7) samples and marker ladder (lane M). ........................... 43
Figure 4.1 Twenty four hours glucose versus xylose yields for different total enzyme loading (33,
16.5 and 8.25 mg/g glucan) saccharifying AFEX treated corn stover. Above the bar, all enzyme
mixtures contain either EG I/βX or EX/βX (Cluster 1) whereas below the bar (Cluster 2) no such
combinations exist. ....................................................................................................................... 57
Figure 4.2 Ternary plots of models developed to predict glucose yields as a function of varying
enzyme loadings upon hydrolysis of AFEX treated corn stover. Expected glucose yield ranges
are denoted by the different colors/patterns in the legend. A.1, A.2 and A.3 are ternary plots of
CBH I, CBH II and EG I, with EX and βX held constant at 0.15 and 0.04, respectively (as
protein mass ratio where sum of all five protein ratios is unity). B.1, B.2 and B.3 are ternary plots
of CBH I, EX and βX, with CBH II and EG I held constant at 0.19 and 0.32 ratio. A.1/B.1,
A.2/B.2 and A.3/B.3 are plots of 7.5, 15 and 30 mg/g glucan enzyme loading, respectively. For
all experiments, βG was loaded at 10% of the total enzyme (CBH I + CBH II + EG I + EX + βX)
mass loading.................................................................................................................................. 65

xii

Figure 4.3 Twenty-four hours glucose (A) and xylose (B) yields for saccharified AFEX-treated
corn stover for varying bG loadings, held at fixed respective optimum ratios of CBH I,CBH II,
EG I, EX and bX for three different total enzyme loadings. ........................................................ 68
Figure 4.4 Glucose (A) and xylose (B) yield for different enzyme cocktails at 24 hours. ........... 70
Figure 5.1 Four-step strategy for screening glycosyl hydrolases and developing novel enzyme
mixtures to maximize digestibility of pretreated lignocellulose. .................................................. 77
Figure 5.2 Percent glucan (blue bar) and xylan (red bar) conversion after 24 hours hydrolysis of
AFEX treated corn stover. Six bacterial enzymes were added together as a mixture at varying pH
and temperature. Each enzyme mixture contains both bacterial cellulases and hemicellulases (4
mg/g glucan each for LC1, LC2, LX1 and LX2; 2 mg/g glucan each for LβX and LβG). .......... 80
Figure 5.3 Percent glucan (blude bar) and xylan (red bar) conversions after 24 hours hydrolysis
of AFEX treated corn stover. Enzyme mixtures used for experimental data sets in panel A (A to
E), panel B (F to M) and panel C (H, O to R) are based on protein compositions listed in Table
5.3.................................................................................................................................................. 84
Figure 5.4 Glucan (X-axis) versus xylan (Y-axis) conversion after 24 hours hydrolysis of AFEX
treated corn stover. Hydrolysis results from three different total enzyme loadings (inclusive of
CBH I + CBH II + EG I + LX 1 + LX 2 + LβX, as listed in Table 5.4) are depicted by green
triangles (30 mg/g glucan), red squares (15 mg/g glucan) and blue diamonds (10 mg/g glucan).
An additional loading of 3, 1.5 and 1 mg/g glucan of βG was supplemented in each case,
respectively. .................................................................................................................................. 93
Figure 5.5 Glucan (X-axis) versus xylan (Y-axis) conversion after 24 hours hydrolysis of AFEX
treated corn stover for varying relative ratios of cellulases (panel I), xylanases (panel II) and βxylosidase (panel III) at three different total enzyme loadings (a, b and c correspond to 10, 15
and 30 mg/g glucan enzyme loading, respectively). ..................................................................... 96
Figure 6.1 Overall conclusions drawn from each step of the process of hemicellulases
optimization in presence of cellulases during hydrolysis of AFEX treated corn stover. ............ 110
Figure 6.2 Glucose (blue bar) and xylose (red bar) yield after 24 h hydrolysis of AFEX-treated
corn stover for different enzyme cocktails. Core enzymes only contain CBH I, CBH II, EG I (4
mg/g glucan each) and βG (2 mg/g glucan). Endoxylanases include LX1, LX2, LX3, LX4, LX5
and LX6 (4 mg/g glucan each). Accessory hemicellulases LArb, LαGl and LβX were loaded at 2
mg/g glucan each. ....................................................................................................................... 112
Figure 6.3 Glucose and xylose yield after 24 h hydrolysis of AFEX-treated corn stover for
different xylanase loadings. LX1 to LX6 were loaded as different combinations (from single
enzyme loading to 5 enzymes added simultaneously). Open symbol denotes no LX3 or LX4
were included in the mixture. Left half filled symbol denotes LX3 was included in the mixture.
Right half filled symbol denotes LX4 was included. Close symbol denotes both LX3 and LX4
were present in the mixture. ........................................................................................................ 113

xiii

Figure 6.4 Glucose (blue) and xylose (red) yield after 24 h hydrolysis of AFEX-treated corn
stover for different ratios of endoxylanases LX3 and LX4 in total enzymes (CBH I, CBH II, EG I,
LX3 and LX4) loading of 20 mg/g glucan. All enzymes were loaded at equi-mass ratio, except
βG, LβX, LαGl and LArb that were additionally supplemented at 2 mg/g glucan each. ........... 115
Figure 6.5 Glucose (blue) and xylose (red) yield after 24 h hydrolysis of AFEX-treated corn
stover for different ratios of LX3 and LX4. 5 mg/glucan of each CBH I, CBH II, EG I and (LX3
+LX4) were added to the hydrolysis. Other enzymes (βG, LβX, LαGl, and LArb) were each
added at 2 mg/g glucan. .............................................................................................................. 117
Figure 6.6 Xylose yield after 24 h hydrolysis of AFEX-treated corn stover in presence of
accessory enzymes. LβX, LArb and LαGl were loaded individually at different protein loadings,
in presence of an excess of the other two enzymes (1 mg/g glucan each). Major cellulases (CBH
I, CBH II and EG I), hemicellulases (LX3 and LX4) were loaded at 5 and 2.5 mg/g glucan each,
respectively. Additional βG (2 mg/g glucan) was added to prevent cellobiose build-up. .......... 118
Figure 6.7 Glucose (A), xylose (B) and total monosaccharide (C) yields after 24 h hydrolysis of
AFEX-treated corn stover for various enzyme cocktails. Accellerase 1000 is a commercial
preparation. Core cellulases contain CBH I, CBH II and EG I at equi-mass loading along with 2
mg/g glucan βG. Xylanases (LX3 and LX4 at equi-mass ratio) to cellulases (CBH I, CBH II and
EG I) ratio is fixed at 1:3. LβX, LαGl and LArb were loaded at 0.6, 0.8 and 0.6 mg/g glucan,
respectively). ............................................................................................................................... 120
Figure 7.1 Separation of individual cellulases by anion exchange chromatography after 20-fold
dilution of AFEX corn stover hydrolyzate supernatant in pH 7.5, 20 mM Tris buffer without
removal of lignocellulose derived UV-absorbing decomposition products. Enzymes were eluted
by applying a linear gradient of 1M NaCl to the column (dotted line; shown as %B on secondary
Y-axis)......................................................................................................................................... 125
Figure 7.2 Two-step high-throughput HT-FPLC method for separation and quantification of
complex cellulase mixtures present in lignocellulosic biomass derived hydrolyzates. High
molecular weight (>50 kDa) cellulases are separated from AFEX pretreated corn stover
hydrolyzate derived UV-absorbing components (e.g., phenolics) by Superdex 75 gel filtration
and then quantified by anion exchange chromatography (IEX). Enzymes were eluted applying a
linear gradient of 1M NaCl (dotted line; shown as %B on secondary Y axis). .......................... 129
Figure 7.3 Binding isotherms for CBH I (A), CBH II (B) and EG I (C) on AFEX pretreated corn
stover. The extent of binding for individual enzymes was measured for single, binary and ternary
cellulase mixtures........................................................................................................................ 135
Figure 7.4 Binding isotherms for CBH I (A), CBH II (B) and EG I (C) on dilute acid pretreated
corn stover. The extent of binding for individual enzymes was measured for single, binary and
ternary cellulase mixtures. .......................................................................................................... 137
Figure 7.5 Binding isotherms for CBH I (A), CBH II (B) and EG I (C) on untreated corn stover.
The extent of binding for individual enzymes was measured for single, binary and ternary
cellulase mixtures........................................................................................................................ 139

xiv

Figure 8.1 Sugar yield and the percentage of enzymes in supernatant for Cellulose I (A),
Cellulose II (B), Cellulose III (C) and amorphous cellulose (D) during 48 hour hydrolysis at 50
ºC and comparison of average hydrolysis rate for different cellulose allomorphs (E). .............. 158
Figure 8.2 The level of bound CBH I, CBH II and EG I for different cellulose allomorphs at 1
hour. ............................................................................................................................................ 161
Figure 8.3 Relative activity for CBH I, CBH II and EG I after certain time of incubation at 50 ºC.
..................................................................................................................................................... 162
Figure 8.4 Sugar yield and the percentage of enzymes in supernatant for AFEX CS with (B) and
without (A) endoxylanase and β-xylosidase during 48 hour hydrolysis at 50 ºC. ...................... 166
Figure 8.5 Sugar yield and the percentage of enzymes in supernatant for ACID CS with (B) and
without (A) endoxylanase and β-xylosidase during 48 hour hydrolysis at 50 ºC. ...................... 168
Figure 8.6 Sugar yield and the percentage of enzymes in supernatant for IL CS with (B) and
without (A) endoxylanase and β-xylosidase during 48 hour hydrolysis at 50 ºC. ...................... 169
Figure 8.7 Percentage of enzyme in supernatant after 48 hours hydrolysis of AFEX CS, ACID
CS and IL CS. ............................................................................................................................. 170

xv

KEY TO SYMBOLS OR ABBREVIATIONS

acetonitrile (ACN)
dilute acid treated corn stover (ACID-CS)
ammonia fiber expansion (AFEX)
p-aminophenyl-1-thio-β-D-cellobioside (APTC)
ammonia recycle percolation (ARP),
β-glucosidase (βG)
β-xylosidase (βX)
carboxymethylcellulose (CMC)
consolidated bio-processing (CBP)
catalytic domain (CD)
cellobiohydrolases (CBHs)
carbohydrate binding domain (CBD)
Consortium for Applied Fundamentals and Innovation (CAFI)
3,5-dinitrosalicylic acid (DNS)
dissolved oxygen (DO)
degree of polymerization (DP)
degree of synergism (DS)
endo-1,4,-β-D-glucanases (EG)

xvi

endoxylanases (EX)
Fungal Genetics Stock Center (FGSC)
Fast Performance Liquid Chromatography (FPLC)
glycosyl hydrolases (GH)
ion exchange chromatography (IEX)
L-arabinofuranosidase (LArb)
α-glucuronidase (LαGl)
Liquid hot water (LHW)
bacterial endoxylanases (LXs)
para-nitrophenyl (pNP)
4-nitrophenyl-β-D-xylopyranoside (pNPX)
4-nitrophenyl-α-L-arabinofuranoside (pNPAf)
4-nitrophenyl-β-D-cellobioside (pNPC)
4-nitrophenyl-β-D-glucopyranoside (pNPG)
Recombinant endo-glucanase (REG)
trifluoroacetic acid (TFA)
yeast nitrogen base (YNB)

xvii

CHAPTER 1 INTRODUCTION
1.1 Background
Depleting crude oil reserves, environmental problems due to green house gas emissions using
fossil fuel and long term energy security have attracted significant attention towards the use of
alternative renewable resources for production of fuels and chemicals [1-3]. Among many types
of biofuels, ethanol is the most employed liquid biofuel [4]. It can be directly used as fuel or
mixed with gasoline at different ratios [5, 6]. Currently, 10% ethanol is mixed into gasoline as an
oxygenate to boost octane number for combustion engines [7]. Slightly modified engines can run
using 85% ethanol (E85) [6]. Almost 90% of Brazilian automotive fuel demand is now met using
ethanol produced from sugar cane [5].
In the US, most of the ethanol is produced from corn starch [4]. Nowadays less than 10% of fuel
demand is met using corn ethanol and there is a growing controversy of producing fuels out of
food materials (about 30% of US corn is used for biofuels production) [8, 9]. Lignocellulosic
11

biomass provides a renewable resource (approximately 1×10

tons) for the sustainable

production of biofuels/biochemicals, and could serve as an important contributor to the world
energy portfolio in the near future [5]. The US has initiated several biofuels research and
development programs that aim to make the cellulosic ethanol cost competitive by 2012 and
replace 30% of the US’s current fuel use by 2030 [10-12].
Although lignocellulosic biomass is considered as a renewable feedstock for biofuels production,
it is quite challenging to convert it to biofuels in an industrial scale. Some of these challenges
include: (i) the bulk density of biomass (agricultural residue) is low and has issues related to

1

logistics on the transportation and storage [13]; (ii) it is highly recalcitrant which makes it harder
to digest by enzyme and requires pretreatment which could be an energy intensive process [1,
14]; (iii) high enzyme requirements add operating cost [1, 15, 16] and (iv) other issues related to
the xylose utilization during fermentation [17, 18], inhibition of enzymes and microbes by
degradation products formed during pretreatment [19-21].
1.2 Objectives
This research applies engineering strategies to optimize enzymatic hydrolysis process of
pretreated biomass as well as to determine some important hydrolysis related parameters which
are required to achieve fundamental understanding of the hydrolysis mechanism. The specific
objectives are:
(1) Purify major biomass degrading enzymes including cellulases and hemicellulases using
standard chromatography procedures.
(2) Optimize the ratio of major enzymes for digesting ammonia fiber expansion (AFEX) treated
corn stover using statistical engineering design.
(3) Study the auxilliary enzymes (in low abundance but do help improve hydrolysis)
performance on AFEX treated corn stover.
(4) Set up the experimental procedures to evaluate enzyme binding properties on biomass.
(5) Investigate the enzyme binding during hydrolysis and evaluate the unproductive binding of
enzyme on lignin.

2

CHAPTER 2 LITERATURE REVIEW
2.1 Lignocellulosic biomass
lignocellulosic
A potential source for fermentable carbohydrates is lignocellulosic materials from plant cell
walls which are composed of up to 75% carbohydrates. Normally, cellulosic biomass contains
40-50% of cellulose, 25-35%, of hemicelluloses, 15-20% of lignin and small amount of minerals,
oils, sugar and other compounds[22] The structure of the cell wall of plants (shown in Figure 2.1)
[22].
shown
is very complicated with cellulose microfibril and hemicelluloses branches.
microfibrils

Figure 2.1 Scale model of the polysaccharides in an Arabidopsis leaf cell. Between
microfibrils, the hemicelluloses cross
cross-links (shown in orange) are extended[23]
[23].
“For interpretation of the references to color in this and all other figures, the reader is
For
referred to the electronic version of this dissertation.”

3

2.1.1

Cellulose

Cellulose, characterized by Anselme Payen in 1838, is the main component in cell walls of
10

higher plants. It is also formed by some algae, fungi, bacteria, etc. About 7.5×10

tons of

cellulose is formed and degraded each year, establishing it as the most abundant regenerated
material on earth[24]. The structure of cellulose molecules (Figure 2.2) shows that cellulose is a
linear polymer consisting of D-glucopyranose joined together by β-1,4 glycosidic bonds with a
degree of polymerization (DP) up to 20,000. Anhydrocellobiose is the repeating unit of
cellulose[24]. The cellulose molecule is very robust because of the hydrogen bonds and linear
crystal structures. The half-times for spontaneous degradation of the glucosidic bond at 25 ºC is
around 5 million years[25]. So enzyme-driven degradation of cellulose is very important to
degrade the cellulose polymers.

4

Figure 2.2 Cellulose structures with cellobiose unit linked through β-1,4-glycosic bond.[26]

5

2.1.2

Hemicellulose

Hemicellulose, the second most abundant polysaccharide in nature (about one
one-third of all
renewable organic carbon on earth), is a major polysaccharide in plant cells[27]. It contains the
cells[27]
following residues: D-glucose, D
glucose, D-galactose, D-mannose, D-xylose, L-arabinose, D-glucuronic
arabinose, D
acid and 4-O-methyl-D-glucuronic acid
glucuronic acid[28]. Xylan is the major constituent of hemicellulose. In
.
addition to xylan, hemicellulose also contain other heteropolymers such as xyloglucan
contains
(heteropolymer of D-xylose and D ucose), glucomannan (heteropolymer of D-glucose and Dxylose
D-glucose),
D
mannose), galactoglucomannan (heteropolymer of D
D-galactose, D-glucose and D-mannose) and
glucose
D
arabinogalactan (heteropolymer of D
D-galactose and arabinose)[29]. Hemicelluloses are
.
connected through hydrogen bonds to cellulose microfibrils and also covalently attached to
lignin to form a highly complex structure.
2.1.3

Lignin

vascular
Lignin is the main component of va cular plants. It is an amorphous, polyphenolic material
composed of three phenylpropanoid monomers: (1) p-coumaryl alcohol, (2) coniferyl alcohol
coumaryl
and (3) sinapyl alcohol[30].

Figure 2.3 Lignin’s three component
components.(1) p-coumaryl alcohol, (2) coniferyl alcohol and (3)
coumaryl
sinapyl alcohol

6

Lignin embeds the cellulose and also forms covalent bonds to hemicelluloses to reduce microbial
and chemical decomposition. Normally, herbaceous plants have a lower content of lignin,
whereas softwoods have higher lignin level[28].
2.2 Pretreatment of biomass
Cellulose can be broken down to glucose either by enzyme hydrolysis or by acid hydrolysis.
Generally, conversion of lignocellulosic substrates to ethanol includes four main steps:
pretreatment, hydrolysis, fermentation and product separation/purification. Pretreatment is the
crucial step which affects the overall biorefinery efficiency[22]. The resistance of lignocellulosic
materials to conversion to fermentable sugar and the high cost of enzymes[31] are two major
processing obstacles. According to the National Renewable Energy Laboratory’s report[32],
pretreatment is one of the most costly steps in cellulosic ethanol production, accounting for 33%
of total processing costs. Hence, successful pretreatment methods not only can increase enzyme
accessibility but also facilitate the downstream hydrolysis and fermentation process.
Although many physical, chemical or biological pretreatment techniques have been studied over
many years, the total cost of pretreatment is still a major obstacle to make price competitive biofuels. So far, only a few pretreatment methods have been proved to be promising for increasing
cellulases digestibility[33] and decreasing cost. Current leading biomass pretreatment
technologies can be categorized into three groups: the acid based pretreatment such as dilute acid
pretreatment and liquid hot water pretreatment, the alkaline based pretreatment including lime
pretreatment, Ammonia recycle percolation (ARP), Ammonia fiber expansion (AFEX) and
extraction based pretreatment such as ethanol organosolv pretreatment.

7

2.2.1

Dilute acid pretreatment

Dilute acid normally refers to 0.5-1.0% sulfuric acid. Although SO2 can enhance the yield in a
way similar to dilute sulfuric acid, it is not preferred from the economics consideration[34]. Acid
or SO2 catalyzed pretreatment can efficiently recover hemicelluloses-derived sugars and improve
cellulosic biomass hydrolysis[35]. Despite little lignin being dissolved, results show that lignin is
disrupted which increased cellulose susceptibility to enzyme. And it has also been shown that
under severe conditions (high temperature, long residence time and high concentration of acid),
nearly complete hydrolysis can be achieved. However, a high proportion of sugar was lost due to
degradation. And degradation products are the main source of inhibitors of downstream
hydrolysis and fermentation[36]. Detoxification techniques such as alkali treatment, sulfite
treatment, anion exchange, enzymatic detoxification and other methods are available to decrease
the inhibition of degradation sugar. However, the multiple steps involved inevitably increase the
whole pretreatment cost. In addition, the corrosion of the equipment by acid solutions is also
considered as an important cost factor.
2.2.2

Liquid hot water pretreatment

Liquid hot water (LHW) plays a role similar to dilute acid at hightemperatures. At 200 °C,
water’s pH is 5.6 compared with pH 7.0 when water is at 25 °C because the pKa of water is
affected by temperature. By LHW pretreatment, acetic and uronic acids are formed by breaking
hemiacetal linkages of hemicelluloses. Those acids catalyze the removal of oligosaccharides
from hemicelluloses[37]. Compared with dilute acid pretreatment, some researchers[38, 39]
believe the LHW pretreatment has the potential to increase cellulose digestibility, sugar yield,
pentosan recovery[22] with the advantage of little or no inhibition of sugar fermentation[40] as

8

well as reducing the need for neutralization. Scale-up LHW treatment at a pilot plant has been
done and hydrolysis cost is estimated equivalent to less than $0.84/gal of ethanol produced from
the corn fiber[41].
2.2.3

Lime pretreatment

Lime (calcium hydroxide) is added to biomass as a pretreatment agent to enhance the enzymatic
digestibility[42]. Approximately 10% of glucan is solublilized after treatment. During lime
treatment, some lignin is removed, which is believed to be one of the reasons to increase biomass
enzyme accessibility. Lime treatment method shows significantly enhanced enzymatic
digestibility when dealing with low-lignin biomass materials, such as switchgrass, bagasse and
wheat straw.
Compared with the NaOH (0.68$/kg)[43] pretreatment method, lime (0.06$/kg)[43] pretreatment
is much cheaper. And the potential to recover lime[44] can also decrease the final cost. If the
operation is employed under 100 °C, no pressure reactors will be required and cost efficiency
will be improved consequently. In addition, lime has less health hazard concerns than some other
pretreatment chemicals. However, the water loading required is more than 5 g H2O/g biomass,
which could be a problem either from the perspective of cost or industrial process burden. Also,
some glucan dissolves in the water and results in the loss of substrate.
2.2.4

Ammonia recycle percolation

Ammonia has several advantages as a pretreatment agent. It is an effective swelling reagent for
lignocellulosic biomass. It has high selectivity for reactions with lignin over with cellulose. And
it is relatively easy to recover due to its highly volatile character. And as an industrially favored
chemical, it is regarded as non-polluting and non-corrosive chemical with only one-fourth the

9

cost of sulfuric acid on molar basis. Kim et al.[45] studied the pretreatment effect on corn stover
using aqueous ammonia in a flow-though column reactor. The corn stover feedstock was
presoaked overnight in 15% NH3, and then followed by the ammonia recycle percolation (APR)
process at 170 °C and 15% of NH3. The results show that the ARP process removed 70%-85%
of the total lignin and that 70% of lignin was removed within 10 min of treatment.
Approximately half of the hemicelluloses were also solubilized. ARP reduced the total solid
mass and the solid remaining was in the range of 53.6%-61.4% of the starting material. Over 95%
of the glucan and nearly 100% for xylan were preserved in the solid. The enzymatic digestibility
at 60 and 10 FPU/g of glucan were near 100% and 92.5% respectively[45].
2.2.5

Ammonia Fiber Expansion

Ammonia fiber explosion (AFEX, now called ammonia fiber expansion) pretreatment was
invented by Dale et al [14, 15, 46, 47]. In AFEX the lignocellulosic biomass was treated with
liquid ammonia at temperatures (60-100 °C) and high pressure (250-300 psi) for 5-10 min.,
followed by rapidly releasing pressure. Both temperature and pressure decreased rapidly. The
surface area available to microbial attack is increased, which was believed to be the primary
mechanism by which AFEX treated biomass achieves the theoretical yields with lower enzyme
loading (<5 FPU per gram of biomass)[48]. Lignin is cleaved and some is deposited on the
surface of the biomass[49]. Compared with other pretreatment methods, AFEX has some unique
characteristics. It can treat biomassthe samples without involving any aqueous solution[50] .The
treatment is relatively rapid and consequently has the potential to get high efficiency treatment in
industrial application. Most of the ammonia can be recovered and used for the next batch of
treatment. Residual ammonia can be used as nitrogen source for downstream fermentation.

10

Because there is no wash stream required, cellulose and hemicelluloses are well preserved with
little degradation product formation[51].
So far, AFEX has been applied to several cellulosic biomass materials such as alfalfa[52],
florigraze rhizoma peanut[48], switchgrass[31], corn stover[53], reed canarygrass[54]. Various
parameters affect AFEX treatment performance including, temperature, ammonia loading,
retention time, particle size[49] and moisture content of biomass.
2.2.6

Ethanol Organosolv

This process extracts most of the lignin from lignocellulosic biomass using an ethanol and water
mixture at around 200 °C and 400 psi[55]. The diluted spent liquor was processed by flashing
and lignin was recovered as a precipitate. The cellulose and some residuals of lignin and
hemicelluloses were retained in the pulp. Enzyme hydrolysis results showed that the lower the
remaining lignin content, the higher cellulose to glucose conversion yield is achieved. The four
independent operation conditions affecting the pretreatment include: temperature, time, catalyst
dose and ethanol concentration[56]. Besides the cellulose substrate for fermentation obtained,
many other co-products are also available after pretreatment. The multi-products refinery plant
has the potential to offset the cost to produce ethanol by producing other materials or chemicals
such as lignin, acetic acid, furfural and so on.
2.3
2.3.1

Enzyme systems
Cellulase

The half-times of the glucosidic bond at 25 ºC is around five million years [25]. So the enzymedriven degradation of cellulose is very important to break the cellulose polymers. The exo-1, 4-βD-glucanase or cellobiohydrolases (CBHs) move along the cellulose chain and release cellobiose

11

or glucose units from the end. CBH I breaks the cellulose from the reducing end [57] and for
CBH II, it breaks from the non-reducing end [58]. However, CBH II is suspected to have some
characteristics typical of endoglucanases [59, 60]. The endo-1,4,-β-D-glucanases (EG) hydrolyze
internal glycosidic bonds randomly inside cellulose chains [61, 62]. 1,4-β-D-glucosidases, which
mainly hydrolyze cellobiose to glucose, also cleave glucose units from cellodextrins with DP up
to six [63].
2.3.2

Hemicellulase

Hemicellulases constitute two major classes of enzymes namely glycoside hydrolases and
carbohydrate esterases. Xylanases (EC 3.2.1.8) break the xylan backbone at β-1,4 bonds [64-66].
Family 10 xylanases cleaves β-1,4 linkages at least one un-substituted xylopyranosyl residue
adjacent to substituted xylopyranolsyl residues from reducing end. Family 11 xylanases only
cleave from at least two un-substituted xylopyranosyl residue [67]. β-Xylosidases (EC 3.2.1.37)
are exo-glycosidases that hydrolyze short xylooligomers into xylose units [64, 65, 68]. βMannanases hydrolyze mannan and release β-1.4-manno groups which can be further hydrolyzed
to mannose by β-mannosidases (EC 3.2.1.25) [29]. The arabinofuranosidase (EC 3.2.1.55)
removes arabinose and α-glucuronidase cleaves the α-1,2-glycosidic bond of the 4-O-methyl-Dglucuronic acid side chain from the xylan backbone [29, 69]. The major esterases are acetyl
xylan esterases (EC 3.1.1.72) and ferulic acid esterases (EC 3.1.1.73), which hydrolyze acetyl
groups on xylose moieties and the ester bond between the arabinose and ferulic acid respectively
[29]. Characterization of enzymes by their substrates sometimes face limitations because some
enzymes have multiple substrate activities. Some xylanases can hydrolyze cellulose [26] and EG
I has xylan degradation activities [70, 71].

12

Table 2.1 Major enzymes and their functions on cell wall polymers.
Enzyme
Cellobiohydrolyase
(CBH I)
Cellobiohydrolyase
(CBH II)

Function
I Break the cellulose chain from the reducing end
and release cellobiose.
II Break the cellulose chain from the non-reducing
end and release cellobiose. Suspected endoactivities
Endo-glucanase (EG )
hydrolyze internal glulosidic bonds randomly
inside cellulose chains. EG I also has significant
xylanase and xyloglucanase activity
β-Glucosidase
Hydrolyze cellodextrins (DP 2-6, cellobiose,
cellotriose, cellotetrose, cellopentose, cellohexose)
into glucose
Endo-xylanase (GH 10) cleaves β-1,4 linkages at least one unsustituted
xylopyranosyl residue adjacent to substituted
xylopyranolsyl residues from reducing end
Endo-xylanase (GH 11) clearves from at least two unsubstituted
xylopyranosyl residue
β-Xylosidase
hydrolyze short xylooligomers into xylose units
β-Mannanases
β-Mannosidases
α-Arabinofuranosidases
α-D-Glucuronidases
Acetyl xylan esterases
Ferulic acid esterases
GH 61 enzymes
Expansin protein
2.3.3

Ref.
[57, 58]
[58, 59,
72]
[61, 62,
70]
[63]

[67]

[67]

[64, 65,
68]
β-1.4-manno [29]

hydrolyze mannan and release
oligomers
Further hydrolyze manno oligomers into mannose
remove arabinose from the xylan backbone
cleave the α-1,2-glycosidic bond of the 4-Omethyl-D-glucuronic acid side chain from the xylan
hydrolyze acetyl groups on xylose moieties
hydrolyze the ester bond between the arabinose and
ferulic acid
No major detectable activity. When adding with
other enzyme, hydrolysis yield is increased
No detectable activity, disrupting the hydrogen
bonds in cellulose, increase cellulase activity

[29]
[29, 69]
[29, 69]
[29]
[29]
[73, 74]
[75, 76]

Other proteins

Some enzymes or proteins are identified to have no specific acidities toward cellulosic substrates,
yet they increase the hydrolysis yield when combined with other cellulases and hemicelluloses.
The newly discovered family GH 61 enzymes have little detectable activity on glucan. However,
supplementation of GH 61 enzymes in cellulases mixture can decrease enzyme loading by 2 fold

13

[73]. The proper functioning of GH 61 enzyme requires a metal ion which is very different from
other fungal enzymes [73]. Other expansin-like proteins have been found to increase the
cellulase activity possibly by disrupting the hydrogen bonds in cellulose [75, 76]. Some
properties of these enzymes are summarized in Table 2.1.
2.4 Enzyme synergism
The term synergy for enzyme hydrolysis is defined as the circumstance in which the amount of
reducing sugar produced when two or more enzymes acting together is greater than the
summation of the individual enzyme acting alone. Enzyme synergism is classified into two
categories (i) one of the enzyme removes the major inhibitors of other enzymes. (e.g., βglucosidase synergism involves the hydrolysis of cellobiose which is CBH I’s inhibitor [16, 77];
endo-xylanases and xylosidases can break xylooligomers into xylose hence remove the inhibition
caused to cellulose [78]). (ii) one of the enzyme can increase the accessibility of other enzymes
on substrates (e.g., endo-exo synergism, a cellulose chain is attacked by endoglucanases by the
random scission and generates more chain ends for cellobiohydrolases; exo-exo synergism, two
exo acting in concert compared to individual activities [79]). Other possible synergies are, for
example, whee hemicellulases remove hemicellulose wrapped around the cellulose micro fibrils
and thereby increase the cellulases accessibility. The fundamental mechanism of synergisms is
not well understood from a molecular basis due to lack of direct observation and debate
regarding the specific action of individual cellulases. Some proposed mechanisms cannot explain
other phenomena such as the lack of synergistic effect among cellulases from different microbial
strains. [80].

14

2.5 Major challenges of enzymatic hydrolysis
Compared with easily hydrolysable carbohydrates (like corn grain starch), lignocellulosic
biomass faces many challenges when used as feedstocks for industrial scale biofuel production.
Compared to corn ethanol production in Table 2.2, the high cost of enzyme, long hydrolysis time,
and low concentration of fermentable sugar in final hydrolyzates as well as the presence of
unfermentable oligosaccharides all impede the industrial scale production of biofuels by using
cellulosic feedstocks.
Table 2.2 Comparison of ethanol produced from corn kernel and cellulosic biomass

Main Substrate
Pretreatment
Enzyme

Corn
Amylose (α-1,4-linkage of
glucose) and amylopectin (α1,6-linkage of glucose)[81]
Mild
α-Amylase
Glucoamylase [4]
90-110ºC, liquefy starch
60-70ºC, saccharification

Saccharification
Temperature
Enzyme dosage
$0.02-0.05/gallon ethanol[82]
and cost
Hydrolysis time
Hours to 1 day [81].

Cellulosic biomass
Cellulose (β-1,4-linkage of glucose)
Hemicellulose, Lignin
Intensive
Cellulases
Hemicellulases
50ºC

20-30 mg/g glucan
$0.72/gallon ethanol[83]
Up to several days
Monomer glucose, xylose, arabinose
at
moderate
concentration,
High
concentration
of
significant amount of unfermentable
Hydrolysate
fermentable sugars (glucose,
oligosaccharides.[18]
Inhibitory
maltose, maltotriose)[82]
degradation product [19]. Insoluble
lignin.
Expected ethanol
Batch 40g/L [18]
Batch 80-100g/L[4]
concentration
SSF 32-35g/L [84]
2.5.1

High cost of commercial enzymes

The most popular commercial enzymes currently available for the biomass hydrolysis are
produced by the submerged fermentation of the fungus T. reesei [85]. Several mutants have been
isolated and the cellulase productivity using the strain has been improved by more than 20 fold

15

over last several decades [86]. In addition, the fermentation conditions have also been optimized
for growth medium including salts, nutrient, surfactant and inducer [87]. All those factors could
affect the enzyme yield as well as the relative ratios of individual enzymes at different pH and
temperature values. The submerged fermentation process requires higher capital cost, including
higher demand for nutrient supplements as well as enzyme inducers [85, 88].
2.5.2

Low activity of enzymes

The Table 2.3 summarizes the specific activity for some enzymes when compared to commercial
cellulases (from BRENDA database, http://www.brenda-enzymes.info/). Regardless of major
difference in substrates and reaction conditions, commercial cellulases are still inefficient when
compared to other enzymes used in various biological processes.
Table 2.3 Specific activity for different enzymes compared to commercial cellulases.
Enzyme
H. sapiens Catalase
Aspergillus awamori glucoamylase
Bacillus sp. α-amylase
B. taurus DNase I (endo)
S. cerevisiae Hexokinase
H. sapiens DNA pol III
(Spezyme CP+ Novozyme 188) on Avicel

Specific activity*
273,800
21,000
5,009
1,090
120
5.3
0.14

*1 µmol product/min/mg enzyme (specified conditions)
The primary reason for this apparent inefficiency is that cell walls are highly recalcitrant. For
cellulose, intra and inter molecule hydrogen bonds lock the cellobiose unit in a tight packed
structure [89]. Substantial energy is required either to peel the glucan chain or to cleave glycosic
bond, thereby resulting in the low enzyme activity [90]. For cell walls, the existence of
hemicellulose and lignin provides another steric hindrance and chemical shield against enzyme
attachment to the cellulose. In other words, the structure of the cell wall is comparable to a

16

concrete structure where one can visualize the cellulose microfibrils as steel rods embedding in
the mix of lignin and hemicellulose acting as cement [91, 92]. Such structures not only protect
the cellulose from cellulases enzymes attack, they also provide the strength to biomass to resist
tough environmental conditions.
2.5.3

Hydrolysis rate decreasing and lower sugar yield at high solid loading

The slowdown in the enzymatic hydrolysis rate with the increasing sugar conversion has been
reported [93, 94]. The mechanism behind the phenomenon is still poorly understood [95]. It is
widely believed that the substrate is becoming less reactive during hydrolysis. However the
reactivity of substrates is difficult to measure [96]. Other proposed reasons include: (i) enzyme
lose activity during hydrolysis [97, 98]; (ii) enzyme bind unproductively [95, 99] to lignin and
(iii) inhibition by degradation product formed during pretreatment and end-product such as
sugars and oligosaccharides [19-21, 100].
Yang and Wyman [101] have claimed that the decline of the hydrolysis rate for the pure
cellulose is not due to decreased substrate activity. By restarting the hydrolysis at different
conversion, almost same initial hydrolysis rate was observed. One possible explanation for such
observations is that enzymes might be getting “stuck” during the hydrolysis process [101].
Similar results were found on dilute acid pretreated wood [96]. However, it is not clear whether
the biomass is still same after extensive washing by salts and other reagents. Hodge et al found
that the high sugar concentration is the major reason for the enzyme inhibition, while acetic acid
(15 g/L) and phenolic compounds (9 g/L) and furans (8 g/L) have limited effect on reaction
kinetics [21]. Kristensen reported that at a high solid loading, the binding efficiency of enzymes
on biomass decreases and thereby results in a low sugar yield [93].

17

2.6 Current progress on enzymatic hydrolysis
2.6.1

Increase substrates digestibility by pretreatment

Different pretreatment technologies for decreasing the recalcitrance of cell walls [102, 103],
increasing the cellulase accessibility and converting the highly crystalline cellulose to more
amorphous structures [104-106] have been studied extensively [15, 35, 103, 107-109]. AFEX is
an alkaline based “dry to dry” (i.e. no separate liquid fraction arises from the pretreatment)
process and the composition of pretreated biomass is almost identical to that of untreated
biomass [15]. In contrast, dilute acid pretreatment can remove a significant amount of
hemicelluloses (60-80%) and some lignin (<5%) [14]. The degree of polymerization (DP) of
cellulose is largely unchanged after AFEX pretreatment (~ 6000-7000 for corn stover), unlike
dilute acid treatment that results in the decrease of the DP by 60-70% [110]. Both of these
thermo chemical pretreatments are believed to increase the cellulose accessibility to cellulases
through various ultra-structural and physicochemical changes that are not currently well
understood [14, 19, 111]. The physiochemical properties of the pretreated biomass strongly
influence the downstream hydrolysis and fermentation processes.
2.6.2

Hydrolysis of pretreated biomass using crude enzyme mixtures

Crude fungal enzymes from commercial sources normally contain many different types of
proteins and enzymes. The available commercial enzymes cocktail for lignocellulosic biomass
hydrolysis normally can be categorized into cellulase, such as Celluclast, Spezyme CP,
Accellerase 1000, CTec, etc.; β-glucosidase, such as Novozyme 188, Accerllerase BG, etc.;
hemicellulase such as Multifect xylanase, HTec, etc.; and pectinase such as Multifect Pectinase.
Mostly, those commercial cocktails contain cross activities on cellulose, hemicellulose and other
intermediate hydrolysis products such as cellobiose, xylobiose. The complexity of commercial
18

enzyme is seen from SDS-PAGE and proteomics analysis (As shown in Figure 2.4 and Table 2.4
PAGE
2
[111]).

Figure 2.4 (A) SDS-PAGE of some commercial enzymes. C: Spezyme CP; G: Novozyme
PAGE
188; X: Multifect Xylanase; P: Multife Pectinase; E: Depol 670L
Multifect

19

Table 2.4 Proteomics data for some commercial enzymes. The abundance of each protein is
reflected by the peptide count number detected during analysis.
GH
family

1
3
3
6a
7a
10
11
5
6
7b
12
45
61a
61b
43
51
54
62
67
74

Enzyme Description
acetyl esterase
acetyl xylan esterase I
acetyl xylan esterase II
Acetyl Xylan Esterases
feruloyl esterase
Feruloyl Esterases
β-glucosidase
β-glucosidase
β-Glucosidases
β-xylosidase
β-Xylosidases
exocellulase II
exocellulase I
Cellobiohydrolases
endoxylanase II
endoxylanase I (all family
11 included)
Endo-xylanases
endoglucanase II
endoglucanase
endoglucanase I
endoglucanase III
endoglucanase V
endoglucanase IV
endoglucanase VII
Endo-glucanases
arabinan arabinosidase
α-arabinofuranosidase
α-arabinofuranosidase
α-arabinofuranosidase
Arabinofuranosidases
α-glucuronosidase
α-Glucuronosidase
xyloglucanase
Xyloglucanases

Spezyme
CP
1.2
0.7
0.2
2
-

Novo
188

4.1
4.1
3
3
10.4
27.5
37.9
1.8

7.7
7.7
1.1
1.1
0.1
0.3
0.4
1.3

0.2
0.2

Multifect Multifect
Xylanase Pectinase
4.4
1
0.4
5.8
1.2
1.2

Depol
670L
0.2
0.2
-

2.9
2.9
6.5
6.5
1
2
3
0.6

4.6
4.6
4
4
0.3
0.8
1
2.6

13
23
36.1
0.4

35.8

1.8

4.1

1.3
0.6

36.4
0.2

4.4
3.1

0.2

0.2
0.4

1.7

4.5
7.6
0.4
4.6
2.8
0.3
0.7

2.6
4.4
5.3
7.5
2.4
0.5
0.2
15.8

0.8
0.5
0.9
1.1

0.9
1
1.9
2.4
2.4
11.3
11.3

2.5

20

0.9
0.1
1.9

1.2
1.2
2.5
2.5
2.5
21.3
21.3

0.2
5.1
3.1
3.6
7.4
2.8
16.9
1.4
1.4
0.7
0.7

2.2
2.2
-

16.5

1.3
1.4
2.7
12
12

Table 2.4 (cont’d)
GH
family
13
15

28

2
5
5
16
17
18
20
27
30
31
32
35
47
53
55
65
71
72
76

Enzyme Description
α-amylase
glucoamylase
Amylases
lyases (all PL families
pectin methyl esterase
rhamnogalacturonan acetyl
α-rhamnosidase
polygalacturonases (endo/
Pectinases
other noncellulolytic
proteins
cip protein 1
cip protein 2
swollenin
β-mannosidase
β-mannase
β-1,6-endogalactanase
GH 16 glycosyl hydrolase
β-1,3-glucanase
chitinase (endo)
chitinase (exo)
α-galactosidase
glucanase,
glucosylamidase,
glucuronoxylanase
α-glucosidase
β-fructofuranosidase
β-galactosidase
mannosidase (in
oligomannosaccharides)
arabinogalactan betagalactosidase
β-1,3-glucanase (endo/exo)
trehalase
α-1,3-glucanase
glycosyl transferase
α-1,6-mannase

Spezyme
CP
0.4
0.4

Novo
188
3.4
40.2
43.6

0.2
0.1
0.1

0.2

2.6

27.4

2.7
3
3.2

Multifect Multifect
Xylanase Pectinase
1.2
0.2
3.3
0.2
4.4
4.2
2.6
0.5
1.5
6.2
15
5.6

20

2.2
2.1
1.7

1.4
0.7

0.8
1.5

2
0.4

0.2
0.3
0.1

1.4
1.3
0.1
1.1
3.5

0.2

0.4

2.3

1
1.1
1.4

2.7
0.3
8.3

6.1
9.5
3
2.5
1.1
1.6
0.2
3.3
0.4

0.1

0.3
0.3
0.4

3.9

0.6
0.7
0.2
0.3
1.8

Depol
670L
0.2
2
2.2
0.5
1.4
1.5

0.4

0.1
0.2

1.4
1.2
0.8

0.3

21

1.3
0.4
0.1
0.6
0.1

1.2

Table 2.4 (cont’d)
GH
family
79
81
92

Enzyme Description
β-glucuronidase
β-1,3-1,4 endoglucanase
α-1,2-mannosidase
Other proteins

Spezyme
CP

Novo
188

16.6

0.5
42.1

Multifect Multifect
Xylanase Pectinase
0.8
0.1
0.5
17.1
44.1

Depol
670L

14.2

To measure those crude enzymes activities, artificial substrates or substrates which were
subjected to significant chemical modifications have been used such as Avicel, xylan,
carboxymethylcellulose (CMC), cellobiose, xylobiose and para-nitrophenol based glycosides
[112, 113]. Activities based on the standard assay have limited ability to predict the real
performance of those enzymes on the whole cell wall or the pretreated biomass [114]. Because
hydrolysis normally operates in days whereas the activity assay normally is done in
minutes/hours, enzymes stability during a long period of hydrolysis is also not reflected. In
addition, the artificial substrates have many differences in physical and chemical structures when
compared to the pretreated cell walls. Many effects such as unproductive binding of enzyme on
lignin, inhibition of degradation products formed during pretreatment and heterogeneous
structure of the cell walls cannot be mimiced using the artificial substrates only [115].
Many other researchers have focused on adding the commercial enzyme cocktail to evaluate
biomass digestibility for a specific pretreatment. Kumar et al. compared the effect of xylanases
supplements on the corn stover pretreated by different pretreatments and found the glucose yield
increased linearly with the remove of xylan for all pretreatments [100]. Some reports compare
different pretreatment technologies with different cellulosic biomass (corn stover, poplar,

22

switchgrass) using commercial crude enzymes, for example the reports by the Consortium for
Applied Fundamentals and Innovation (CAFI) group [116-124].
2.6.3

Hydrolysis studies by using purified enzymes

Many researchers use artificial pure cellulose substrates, such as Avicel, CMC, filter paper,
swollen cellulose. Irwin et al. measured the activity of the purified T fusca cellulases in the
presence of T reesei enzymes (CBH I, CBH II). Addition of CBH I to T. fusca crude cellulase
increased 1.7 fold itsactivity toward filter paper. Wood et al. found the optimal ratio of CBH I
and CBH II was 1:1 and trace amount of endoglucanase activity enhanced CBHs mixture
effective on hydrolyzing cotton [62]. Baker et al. compared the performance of 5 ternary enzyme
cocktails of cellulase from different strains and found that the CBH I from T. ressei, an
endoglucanases from A.cellulolyticus, and an exo cellulase delivered high hydrolysis rates over a
wide range of mixture compositions on microcrystalline cellulose [125]. But the highest yield is
less than 20% due to the low enzyme loading range selected by the authors. Similarly, Kit et al.
optimized 6 cellulases mixtures on the filter paper and observed significant synergism among
those cellulases [126]. The degree of synergism (DS), [127] which is defined as the activity
exhibited by mixtures of enzyme components divided by the sum of the activities exhibited
individually by the enzymes, is commonly used by many researchers. DS values larger than two
are very common and in some cases, the DS was larger than five [128]. The DS is closely related
to and affected by the dosage of the enzyme loading, characteristics of substrates, extent of
hydrolysis, substrate concentration, etc [129, 130]. Hence it is difficult to compare DS from
different researchers. Boiset et al. found the mixture of enzymes giving rise to the highest
saccharification rate did not always correspond to mixtures with highest DS [131]. Therefore,
from the engineering point of view, it is preferable to use the hydrolysis yield to evaluate the

23

hydrolysis. Hydrolysis yield not only reflects the extent of hydrolysis but also the available sugar
for the downstream fermentation. In addition, the yield is an important factor for determining the
capital and process cost in the large scale biofuel production.
The native T. ressei normally produces 40-60% CBH I, 12-20% CBH II, 5-10% EG I and 10-30%
of other enzymes [132, 133]. This ratio indicates substrate-specific gene regulation and response
in Trichoderma strain [132]. These enzyme cocktails may not work well for all pretreated
biomass. Therefore, the ratio optimization of an individual enzyme for a specific pretreated
biomass has an obvious engineering significance. The optimized cocktail can reduce the enzyme
loading without sacrificing the hydrolysis yield or increasing the sugar yield in shorter
incubation time. Eventually, it could help construct multi gene expression systems in fungi to
produce optimized enzyme mixtures [134] or help more rationally blend crude enzyme mixtures
from different strains. In addition, it helps to determine the critical enzyme therefore rationally
design heterogeneous expression of the enzyme during consolidated bio-processing (CBP) [32,
135-137].
For the ratio optimization applied on pretreated biomass, Rosgaard et al. optimized CBH I, II,
EG I, II on barley straw with different pretreatment methods[71]. The steam explosion and liquid
hot water pretreated straw had the similar optimum ratio of CBH I, CBH II and EG I
(0.43:0.20:0.37) while EG II was not required. If the biomass is first soaked with acid and then
steam explosion pretreatment is performed, the EG I’s ratio decreased to 0.27 from 0.37. The
acid soaking hydrolyzes a part of the xylan and EG I has a significant activity on xylan
hydrolysis [71]. Partial removal of xylan helps to reduce EG I and also facilitates the improved
cellulose hydrolysis (20% yield increased compared with other two pretreatmented straw) [138].
Kim et al. optimized the five Thermomonaspora fusca cellulase with or without CBH I from T

24

reesei and found the enzyme cocktail with CBH I exhibited three times the yield when compared
to non-CBH I contained cocktail. The optimized enzyme cocktail required 43.3% of CBH I in
total enzymes [126]. Selig et al. found significant increases in the depolymerization of the liquid
hot water pretreated corn stover by adding purified xylanase and esterases to CBH I. Xylanase
and esterase work synergistically on the xylan hydrolysis and in turn improve the cellulose
hydrolysis [139]. Zhou et al. optimized purified enzymes from Trichoderma viride on the steamexploded corn stover and found the optimized relative ratio for CBH, EG and βG was 62.0%,
35.2% and 2.8% respectively which increased the glucose yield by 2.1 folds to 72.4% when
compared to crude cellulase enzyme preparation [140]. Benko et al. reported that xyloglucanase
(EG I, EG III and Cel74A xyloglucanase) can increase the hydrolysis yield on 11 different
pretreated biomass with the combination of purified CBH I, II, EG II and βG [70].
2.6.4

Enzyme binding characteristics

Cellulase typically contains a catalytic domain (CD) and a carbohydrate binding domain (CBD)
[141] joined by an extended interdomain linker peptide [142]. The extent of binding and
processive hydrolytic action of CBHs on crystalline cellulose depends on both the CBD and CD
[57, 142, 143]. CBDs are thought to enhance the CD hydrolysis efficiency on the insoluble
substrates via increasing the local surface bound enzyme concentrations [141, 142, 144]. Binding
of the cellulase enzymes precedes their hydrolytic actions. Therefore, it is critical to obtain a
better understanding of the binding properties of individual cellulases on pretreated
lignocellulosic biomass.
Currently, most cellulase binding studies fall into three categories: 1) quantification of the
enzymes present in the supernatant using electrophoresis [145, 146]; 2) monitoring the modified
enzymes with fluorescent tags [33, 147] or isotope labels [148, 149]; and 3) measuring the total

25

protein concentrations [150-153] or cellulase activity [154, 155] remaining in supernatant. The
characteristic advantages and limitations of each technique are listed in Table 2.5.
Electrophoresis based quantification method is a simple technique with reproducibility problems
which make it a semi-quantitative method for proteins that are able to be separated via
electrophoresis. Fluorescent labeling methods are typically suitable for pure cellulosic substrates
(e.g. Avicel, phosphoric acid swollen cotton, bacterial cellulose). Lignocellulosic biomass
normally exhibits strong auto-fluorescence which can overwhelm the protein signal [33]. In
addition, various labeling procedures can modify the native protein structures and thereby affect
their binding properties. Protein radio-labeling, which requires specialized equipment, is labor
intensive and hence is not widely used [148, 149]. Methods that measure total protein using
bicinchoninic acid or other chemical reagents are unable to quantify individual enzymes in
complex enzyme mixtures and are also prone to interference from reducing sugars, lignin
phenolics and other non-hydrolytic proteins present within cell walls.

26

Table 2.5 Currently available cellulase adsorption measurement methodologies
Method

Main principle

SDSPAGE

Enzyme
concentration by
relative band
density on gel

Label enzymes
Fluorescent with fluorescent
labeling
groups for
quantification

Isotope
labeling

Total
protein

Enzyme
activity

FPLC

Culture strains on
isotope labeled
media and purify
expressed
enzymes
Measuring total
nitrogen, or using
Bradford or BCA
reagents to
quantify total
protein
concentration
Measuring
supernatant
enzyme activities
to correlate to
enzyme
concentration
Using ionexchange column
to quantify
individual
enzymes with
different pI by
correlating to UV
absorption peak
area.

Advantages
Easy to operate,
minimal
interference by nonproteinaceous
compounds
Simulataneous
quantification of
multiple enzymes
by labeling different
emission
wavelength
fluorescent markers

Limitation
Semi-quantitative,
Not applicable to
proteins with
similar molecular
weight
Fluorescence
bleaching,
interference due to
lignin/phenolic
autofluorescence,
risk modifying
enzyme binding

Quantify individual
enzyme in mixture

Labor intensive,
special equipment
necessary

Quick, simple,
inexpensive
measurement
without need for
sophisticated
labware

References
[145, 146]

Cannot differentiate
between enzymes,
interference by
background sugars
and phenolics

[33, 147]

[148, 149]

[150-153]

[154, 155]
Easy to assay,
minimal
interference by nonenzymatic proteins.

Lack of suitable
substrates with
enzyme specificity,

Quantify individual
enzymes, accurate,
minimal
interference by
background sugars
and salts.

Low throughput and
laborious,
interference by UV
absorbing
compounds, applied
to two enzyme
based mixtures

[79, 156]

27

Hence, developing a robust analytical method that can quantify individual cellulases present
within lignocellulosic hydrolyzates is critical to improve our understanding of the enzyme
synergism, productive/unproductive enzyme binding and the role of pretreatment on the enzyme
accessibility to lignocellulosic plant cell walls.
Fast Performance Liquid Chromatography (FPLC) based separation and quantification of
proteins has been used previously by Medve et al. for CBH I/CBH II [156] and CBH I/EG II [79]
mixtures hydrolyzing microcrystalline cellulose. Separation of these proteins is possible due to
differences in their isoelectric points (pI). Enzymes bound on ion-exchange columns can be
eluted separately by linear salt gradients. Pretreated cell walls have additional UV absorbing
compounds (e.g. aldehydes, phenolics) that can interfere with the separation and quantification
of enzymes [19].
2.6.5

Others

Other research to improve the hydrolysis efficiency include using genetic engineering tools to
increase the yield of biomass or modify other characteristics of the biomass in favor of the fuel
production [157], as well as modified lignin structures to the facilitate pretreatment [102, 158].
Protein engineering has been used to increase individual enzyme’s stability and activity [159].
CHAPTER 3 GLYCOSYL HYDROLASES EXPRESSION AND PURIFICATION
3.1 Introduction
To our knowledge there have been few reports of the production of biomass hydrolyzing
enzymes by culture of recombinant Pichia pastoris although several industrial enzymes have
been produced from this yeast [160]. The recombinant cellulases produced by fermentation of P.
pastoris were tested for their ability to act in synergism with purified commercial enzymes. An

28

advantage of using heterologous expression for protein isolation is to minimize the purification
steps necessary to achieve high enzyme purity.
Fast-flow Protein Liquid Chromatography (FPLC) based methods have been used to purify
cellulase enzyme components from Trichoderma viride [161], Pennicillium brasilianum [162],
Aspergillus sydowii [163], and Trichoderma reesei [164]. It has been reported that CBH II from
Trichoderma reesei is difficult to purify from enzyme mixtures due to the tendency of CBH II
and EG’s to form aggregates in solution[165]. To separate CBH from EG, p-aminophenyl βcellobioside has been used as an effective affinity ligand to capture CBH[166]. However, this
affinity resin is expensive, requires a hydrogenation reaction under platinum catalyst for
synthesis and is not available commercially. Trace contamination of endoglucanases appearing in
purified CBH’s could result in confounded results during activity assays. Researchers have
observed purified CBH’s with very low CMCase activity, possibly due to endoglucanase
contamination, resulting in conflicting reports [62, 164, 167]. Therefore, it is imperative to
develop high-throughput purification methods that isolate high purity proteins for enzymesynergy investigations. In this study, common high performance ion exchange and hydrophobic
interaction based chromatographic columns were used to isolate electrophoretically pure
cellulases and hemicellulases from commercial enzymes and culture broths of heterologously
expressing recombinant yeasts.
3.2 Enzyme production
3.2.1

Organism

Recombinant P. pastoris strains containing cellulase and xylanase genes were obtained from the
Fungal Genetics Stock Center (FGSC) at the University of Missouri (Kansas City, MO). The
genes encoding cell wall degrading enzymes were isolated from Aspergillus nidulans and

29

integrated into the genome of P. pastoris X-33 by Bauer et al. (2006) [168]. The recombinant
strains used were FGSC#10062 and FGSC#10077 expressing endo-glucanase (REG) and βxylosidase (βX), respectively. The recombinant strains were maintained on YPD plates
containing yeast extract (1 % w/v), peptone (2 % w/v), dextrose (2 % w/v) and agar (2 % w/v).
o

Plate cultures were stored at 4 C for routine use. Culture stocks in 40 % glycerol were stored at
o

-80 C for long term use.

3.2.2

Culture media

For preparation of seed culture BMGY medium was used. The BMGY medium contained (in 1L)
10 g of yeast extract, 20 g of peptone, 100 ml of yeast nitrogen base (YNB) (20.4 g of yeast
nitrogen base without ammonium sulfate and amino acids and 60 g of ammonium sulfate in 600
ml water), 100 ml of 1M potassium phosphate buffer at pH 6.0, 100 ml of 10 % (v/v) glycerol
and 2 ml of 0.02 % (w/v) biotin. YNB and biotin were filter sterilized and added to the sterile
medium containing other components. For fermentations, rich media BMMY or synthetic basal
salt medium were used. The BMMY medium was composed of yeast extract, peptone, yeast
nitrogen base, phosphate buffer and biotin at concentrations similar to BMGY medium. Instead
of glycerol, 5 ml of methanol was added as the carbon source. The basal salt medium contained
phosphoric acid (26.7 ml), calcium sulfate (0.93 g), potassium sulfate (18.2 g), magnesium
sulfate heptahydrate (14.9 g), potassium hydroxide (4.13 g), glycerol (40.0 g) and 1.4 ml of trace
salts solution in a liter of distilled water. The trace salts solution was prepared by dissolving
CuSO4-5H2O (6 g), NaI (0.08 g), Mn SO4-H2O (3.0 g), NaMoO4-2H2O (0.2 g), boric acid (0.02
g), CoCl2 (0.5 g) ZnCl (20.0 g) FeSO4-7H2O (65.0 g), biotin (0.2 g), H2SO4 (5.0 ml) in one liter
of distilled water. The trace salts solution was filter sterilized using a 0.2 µm membrane.

30

3.2.3

Preparation of seed culture

A single colony from YPD plate was transferred to 5 ml YPD broth in test tubes and incubated at
o

28 C for 24 h with agitation (225 rpm). One ml of the culture was then transferred into 500 ml
o

baffled flasks containing 100 ml of BMGY medium and incubated at 28 C for 24-36 h with
agitation at 225 rpm. The culture broth was centrifuged at 3000 rpm at room temperature for 3
min and the supernatant was discarded. The cell pellet was re-suspended in fresh sterile medium
and used to inoculate production medium for enzyme expression in methanol medium.
3.2.4

Expression of recombinant enzymes in shake flasks

The expression of recombinant enzymes was tested by cultivating P. pastoris in BMMY medium
in shake flasks. Baffled Erlenmeyer flasks (1000 ml) with 200 ml of production medium were
o

inoculated with seed culture to an initial OD600 of approximately 1.0 and incubated at 28 C with
agitation at 225 rpm. The cultures were supplemented with 0.6 % methanol at 24 h intervals for
120 h.
3.2.5

Fermentation

A lab scale 1L BIOSTAT B plus fermentor (Sartorius AG, Goettingen, Germany) was used for
fed-batch fermentation. Cultivation of P. pastoris in batch phase is done for 24 h in glycerol
medium. Then fed-batch fermentation was performed by adding methanol to induce the
expression of recombinant protein. The batch fermentation phase was carried out using either
rich BMMY medium or basal salt medium with 4% (w/v) glycerol. The bioreactor vessel was
filled with 600 ml of the production medium and sterilized in an autoclave. The medium was
inoculated with seed culture to an initial OD600 of about 1.0. The culture pH and temperature

31

o

were maintained at pH 6 and 30 C. pH was adjusted using ammonium hydroxide (28%) which
also served as nitrogen source during fermentation. The level of dissolved oxygen (DO)
concentration was maintained over 20% throughout the fermentation with air and agitation
controls. Anti-foaming agent was added at the start of batch culture to minimize foam formation.
There was no foam formation during 120 h fermentation. The fed-batch fermentation was started
after 24 h of fermentation. Methanol was added to the culture as an inducer for expression of
recombinant enzyme. Addition of methanol was dependent on change in DO level, which was
controlled by a multistage algorithm encoded into an Excel spreadsheet. Initially, the stirring rate
was increased to maintain 20% DO. Once the stirring rate reached the maximum (900 rpm),
oxygen was supplied to maintain the DO level. Methanol feeding was based on substrate
depletion which was identified by a spike in the DO level. For basal salt medium based
fermentation, the methanol was mixed with the trace element solution and used for fed batch
additions. In the case of rich medium pure methanol was used in fed batch additions. The fedo

batch fermentation was carried out for 120 h at pH 6 and 30 C. During the fermentation culture,
samples were removed at 24 h intervals and OD600 was measured. After removing the cells by
centrifugation the supernatant was analyzed for expression of recombinant enzyme by activity
assay and SDS-PAGE. At the end of fed-batch fermentation the cells were harvested and the wet
weight of the cell pellet was measured.
3.2.6

Production of recombinant enzymes from P. pastoris

P. pastoris has been used for high level expression of heterologous proteins by employing a
methanol inducible promoter [169]. Several recombinant enzymes and functional proteins such
as lipase and human interferon have been successfully produced at high levels using the P.

32

pastoris system [160, 170]. However, the expression of hydrolases by P. pastoris for degradation
of plant cell walls is not well known. The major advantage of using P. pastoris for glycosyl
hydrolase expression is that individual enzymes of endo-, exo-acting and de-branching activity
for degradation of complex biomass structure can be produced and purified relatively easily. In
recombinant P. pastoris the target gene is placed under the control of methanol inducible alcohol
oxidase 1 promoter (AOX1p) and integrated into its genome. Though a few commercial enzymes
are available for biomass hydrolysis, the actual ratio of individual enzymes in the mixture
required for efficient pretreated biomass hydrolysis has not yet been defined. This is partly due to
inadequate knowledge of the precise composition of enzyme activities in crude commercial
enzyme blends. Here, the P. pastoris expression system was used for production and purification
of major glycosyl hydrolases (GH). The main biomass depolymerizing enzymes endo-glucanase,
β-glucosidase and β-xylosidase were expressed using P. pastoris methanol induction system and
purified. Recombinant P. pastoris strains containing the genes encoding the GH of Aspergillus
nidulans were obtained from FGSC and used for fermentation. The recombinant enzymes were
fused to 6xHis tag at N-terminus for simplified purification of recombinant enzymes [168].
All three enzymes were produced by fermentation using rich BMMY medium. After 24 h of
batch fermentation in glycerol medium the OD600 of cultures reached about 50 which is 50 fold
higher than the initial cell density. Analysis of the culture sample showed absence of
recombinant enzyme expression. The onset of fed-batch culture with the addition of methanol
caused expression of recombinant enzymes. The level of enzyme expression increased with
fermentation time and the maximum level was obtained at 96 h (Figure 3.1). As methanol was
used as the carbon source there was an increase in cell density during fed-batch phase. At the end
of 120 h of fermentation the wet-weight of cells reached 100 to 150 g/L of culture medium. The

33

amount of extracellular protein produced reached about 0.7 – 1.0 g/L. As seen in the Fig. 1 the
recombinant enzyme was the major protein. These results demonstrate the potential of the P.
patoris system for high level expression of other accessory enzymes for efficient hydrolysis of
biomass.

Figure 3.1 SDS-PAGE of expressed recombinant enzymes by P. pastoris during the course
of fermentation. Each lane refers to culture supernatants of different fermentation times in
hours. Each well was loaded with 10 µl of respective supernatant. Where; A Engoglucanase, B - β-Glucosidase, C - β-Xylosidase. Arrows indicate respective enzymes of
interest.
3.2.7

Purification of recombinant enzymes

Since the recombinant enzymes are fused with 6x His tag, a Ni-affinity column was used to
purify enzymes. With low volume (4-5 ml) Ni-affinity column all the recombinant enzymes were
electrophoretically purified. But large scale purification of enzymes from fermentation broth of
cell culture using pre-packed FPLC based Ni-column was not successful. With the help of high
throughput ion exchange column (Resource Q) we could rapidly purify all enzymes. These
results demonstrated the use of P. pastoris for high level expression of individual biomass
hydrolyzing enzymes and one step purification using fast flow ion exchange chromatography.

34

Recombinant endo-glucanase (REG) was produced by fermentation using basal salt medium to
test the expression level and its effect on the purification method. The expression of endoglucanase was at high level similar to rich medium and purification resulted in very pure enzyme.
The fermentation strategy using synthetic medium has good potential to produce and then
efficiently purify other biomass hydrolyzing enzymes. Compared to purification of enzymes
from commercial enzyme source, the purification of recombinant enzymes was simple and
efficient. Since purification of enzymes is a key limiting step for investigating enzyme synergy,
these results are useful for purifying the spectrum of biomass hydrolyzing enzymes from the
crude commercial enzymes and fermentation broth of recombinant P. pastoris. The purified βglucosidase from recombinant fermentation was found to have no activity on cellobiose. The
reasons are unknown.
3.3 Protein purification
Details of enzyme purification steps are shown in Table 3.1. Enzyme purification was performed
using a FPLC system (GE Healthcare, Buckinghamshire, United Kingdom). The following FPLC
columns were used: 51 ml HisPrep 26/10 desalting column (GE Healthcare, Lot # 17-5087-01), 6
ml Resource Q anion exchange column (GE Healthcare, Lot # 17-1179-01), 1.7 ml Mono S
cation exchange column (GE Healthcare, Lot # 17-5180-01), 1.7 ml Mono Q anion exchange
column (GE Healthcare, Lot # 17-5179-01) and 1 ml PHE hydrophobic interaction column (GE
Healthcare, Lot # 17-1186-01). The crude enzyme samples were filtered (using 0.2 µm filter) and
buffer exchanged to initial buffer (buffer A) using HisPrep 26/10 desalting column before
injecting onto respective columns. CBH I and CBH II isolated from Spezyme CP (after steps 4.3
and 2.3) were further polished using APTC (p-aminophenyl-1-thio-β-D-cellobioside) based
affinity chromatography to remove minor endoglucanase contaminants [166]. Protein samples

35

were concentrated using a tangential flow-filtration system (10 kDa Vivaflow membrane, Lot #
08VF5022, Sartorius, Bohemia, NY). Milli-Q water was used to prepare buffers. Elution buffers
were filtered through 0.2 µm PES membrane and degassed prior to use.

36

Table 3.1 Chromatographic steps employed during FPLC based purification of various glycosyl hydrolases from crude
enzyme mixtures.
Step
1.0

2.1

2.2

2.3

3.1

3.2

3.3

4.1

4.2

Sample
Spezyme CP

Column and Buffer
6 ml Resource Q
A: 20 mM Tris pH 7.3
B: A+1M NaCl
CBH II rich fraction in 1.7 ml Mono Q
1.0
A: 20 mM Tris Buffer pH 7.5
B: A+1M NaCl
Major Peak in 2.1
1.7 ml Mono S
A: 20 mM Citric Buffer pH 3.1
B: A+1M NaCl
Major Peak in 2.2
1 ml Resource PHE
A: 20 mM Tris pH 7.5
B: 1M (NH4)2SO4 +A
EG I rich fraction in 1.7 ml Mono Q
1.0
A: 20 mM Tris Buffer pH 7.5
B: A+1M NaCl
Major Peak in 3.1
1.7 ml Mono S
A: 20 mM Citric Buffer pH 3.1
B: A+1M NaCl
Major Peak in 3.2
1 ml Resource PHE
A: 20 mM Tris pH 7.5
B: 1M (NH4)2SO4 +A
CBH I rich fraction in 1.7 ml Mono Q
1.0
A: 20 mM Tris Buffer pH 7.5
B: A+1M NaCl
Major Peak in 4.1
1.7 ml Mono S
A: 20 mM Citric Buffer pH 3.1
B: A+1M NaCl

37

Gradient
2 ml sample
15 CV 0-30% B

Flow (ml/min)
6

10 ml sample
10 CV 0-9% B

2

11 ml sample
25 CV 0-15% B

2

11 ml sample
40 CV 25%-0 B

4

10 ml sample
20 CV 0-13% B

2

10 ml sample
12 CV 0-8% B

2

11 ml sample
40 CV 25%-0 B

4

5 ml sample
20 CV 20-30% B

2

5 ml sample
10 CV 0-10% B

2

Table 3.1 (cont’d)
4.3

Major Peak in 4.2

5.1

Novozyme 188

5.2

βG rich fraction in 5.1

5.3

Major Peak in 5.2

6.1

Multifect Xylanase

6.2

EX rich fraction in 6.1

7

β-xylosidase
fermentation broth
from recombinant
Pichia pastoris FGSC
strain# 10077

1 ml Resource PHE
A: 20 mM Tris pH 7.5
B: 1M (NH4)2SO4 +A
6 ml Resource Q
A: 20 mM Tris pH 7.5
B: A+1M NaCl
1.7 ml Mono S
A: 20 mM Citric Buffer pH 3.1
B: A+1M NaCl
1.7 ml Mono Q
A: 20 mM Tris Buffer pH 7.5
B: A+1M NaCl
6 ml Resource Q
A: 20 mM Piperazine pH 10.6
B: A+1M NaCl
1.7 ml Mono S
A: 20 mM Citric Buffer pH 3.1
B: A+1M NaCl
6 ml Resource Q
A: 20 mM Tris pH 7.5
B: A+1M NaCl

38

5 ml sample
6 CV 55%-0 B

4

2 ml sample
1 CV 20% B
4 CV 20%-44%B
2 ml sample
10 CV 0-100% B

6

2

5 ml sample
20 CV 20-40% B

2

2 ml sample
10 CV 0-50% B

6

2 ml sample
15 CV 0-20% B

2

2 ml sample
15 CV 0-30% B

6

3.3.1

SDS-PAGE and protein purity quantification

SDS-PAGE was performed using a Novex

®

TM

XCell SureLock
®

Carlsbad, CA, USA) using pre-cast NuPAGE

Mini-Cell system (Invitrogen,

Novex 4-12% Bis-Tris gels (Invitrogen, Lot #

NP0321BOX). After electrophoresis the gels were fixed with 50% methanol and 7% acetic acid
solution for 15 min and stained with GelCode Blue Stain Reagent (Thermo Fisher Scientific, Lot
# KD131759, Rockford, IL, USA) to visualize the protein bands. The gel image was taken using
the UVP BioDoc-It Imaging System (Upland, CA, USA). Protein purity was estimated by UNSCAN-IT gel software (Version 6.1, Orem, Utah, USA).
3.3.2

Purification of glycosyl hydrolases from crude enzyme blends

The commercial enzyme blend, Spezyme CP, which has high activity on cellulose, was used as a
source of EG and CBH. Purification of CBH I, CBH II and EG I was performed using suitable
ion exchange columns. The crude enzyme sample was loaded onto the column, pre-equilibrated
with 20 mM Tris buffer at pH 7.3. The bound proteins were eluted using a linear gradient of
NaCl (0 to 1 M) in the same buffer. Four major protein peaks, I, II, III and IV were observed
(Figure 3.2). The fractions corresponding to peaks I and IV had higher Avicelase activity,
whereas Peak II had higher CMC activity (data not shown). Electrophoretically pure proteins
were obtained by further polishing each fraction using higher resolution ion exchange (i.e. Mono
Q and Mono S) and hydrophobic interaction chromatography (Resource PHE) based
chromatography. CBH I and CBH II were further polished using APTC based affinity
chromatography (after steps 4.3 and 2.3, respectively, as indicated in Table 3.1) to remove trace
endoglucanase contaminants. The polished fractions showed single protein bands on SDS-PAGE

39

(Figure 3.5). The degree of purity was found to be >99 % based on quantification of the SDSPAGE gel band intensity using UN-SCAN-IT gel

1,000

TM

software.

120

UV 280

IV

B%

100
80

600
60
400

I
II

B%

UV 280 (mAu)

800

40

III

200

20

0

0
0

50
Elution (ml)

100

Figure 3.2 Separation of protein fractions (I-IV) from commercial enzyme blend (Spezyme
CP) by anion exchange chromatography (Step 1.0) with respect to elution buffer gradient (%
B).
Complete hydrolysis of cellulosic biomass necessitates avoiding inhibition of CBH’s by
cellobiose and other gluco-oligomers [171, 172]. Novozyme 188 was chosen as the source for
purification of β-glucosidase. IEX chromatographic separation gave four major protein peaks
(Figure 3.3), among which peak II gave the highest β-glucosidase activity (based on pNPG and
cellobiose based activity assays). SDS-PAGE analysis revealed two major proteins in Peak II
fraction (data not shown). This protein fraction was further purified (to 94% purity) using a

40

cation exchange column (Mono S). A third step of polishing with Mono Q further increased the
purity to greater than 99% (Figure 3.5).

1,600
B%

100

1,200
1,000

80

800

60

600

B%

UV 280 (mAu)

1,400

120

III

UV 280

40

400

I

IV

II

20

200
0

0
0

20

40
Elution (ml)

60

Figure 3.3 Separation of protein fractions (I-IV) from commercial enzyme blend
(Novozyme 188) by anion exchange chromatography (Step 5.1) with respect to elution
buffer gradient (% B).
Multifect® Xylanase was the source to isolate a suitable endo-xylanase. The separation was
performed using the anion exchange column at pH 10.6 (20 mM Piperazine). Four major peaks
(Figure 3.4) were obtained within which peak II gave the highest xylanase activity. Peak II
fraction was further polished using a cation exchange (Mono S) column at pH 3.1, to obtain high
purity (>99 %) endo-xylanase giving a single protein band on SDS-PAGE.

41

160
140

B%

120

120

I

100

100

80
II

80

IV

60

B%

UV 280 (mAu)

III

UV 280

60
40
40
20

20

0

0
0

50
Elution (ml)

100

Figure 3.4 Separation of endo-xylanase from Multifect Xylanase (Step 6.1). Elution profile
of protein UV adsorption (280 nm) with respect to elution buffer gradient.

42

Figure 3.5 SDS-PAGE of purified EG I (lane 1), CBH II (lane 2), CBH I (lane 3), β-G (lane
4), EX (lane 5), β-X (lane 6), REG (lane 7) samples and marker ladder (lane M).

43

CHAPTER 4 MIXTURE OPTIMIZATION OF SIX CORE GLYCOSYL HYDROLASES
FOR MAXIMIZING SACCHARIFICATION AFEX PRETREATED CORN STOVER
4.1 Introduction
From previous chapter, the most important cellulases and hemicellulases necessary to digest
pretreated biomass have been purified. EG randomly hydrolyzes internal glycosidic bonds within
cellulosic microfibrils [173], while CBH enzymes act processively along cellulosic chains
cleaving off cellobiose units from either end (CBH I acts at reducing ends and CBH II acts at
non-reducing ends) [174] with βG ultimately hydrolyzing cellodextrins to glucose [175]. EX
cleaves the xylan backbone at internal β-1,4 xylosidic bonds, while βX hydrolyzes short
xylooligomers to xylose [29]. All these enzymes are thought to work harmoniously, creating new
accessible adsorption sites or active substrates for each other to act upon [16].
In this chapter, six core cellulases and hemicellulases were isolated using various purification
and heterologous expression strategies. Various combinations of these enzymes were tested on
AFEX treated corn stover to determine optimal combinations at three total protein loadings (8.25,
16.5 and 33 mg/g glucan) using a suitable design of experiments methodology. Synergistic
interactions among different enzymes were then determined through various mixture
optimization experiments. Optimum combinations were predicted from suitable statistical
models which were able to further increase hydrolysis yields. These results demonstrate the
potential to rationally design an enzyme mixture targeted towards a particular feedstock and
pretreatment that can help maximize hydrolysis yields and minimize enzyme usage in a
cellulosic biorefinery.

44

4.2 Materials and Methods
4.2.1

AFEX pretreatment

The detailed procedures of AFEX pretreatment have been described[176]. Pre-milled (passed
through a 10 mm sieve) corn stover (Pioneer Hybrid seed variety (33A14) based stover, provided
by NREL, was harvested in 2002 from the Kramer farm in Wray, CO) with 60% moisture (kg
water/kg dry biomass), was transferred to a high-pressure Parr reactor. Heated liquid ammonia (1
kg of ammonia/kg of dry biomass) was charged to the reactor vessel resulting in immediate rise
o

o

in temperature to 130 C. The reactor was maintained at 130 C for 15 min through an external
heating mantle (within + 10 °C). At the end of 15 min, the pressure was reduced to atmospheric
level resulting in precipitous drop in temperature of the reactor contents. The very rapid pressure
o

drop in the vessel caused the ammonia to vaporize, cooling the biomass to below 30 C. The
pretreated material was left under the hood overnight to ensure complete removal of residual
ammonia. The AFEX treated stover was then milled to under 100 µm based on the methodology
employed earlier [177] and kept under refrigeration until further use. The composition of the
milled AFEX corn stover was found to be 34.4% glucan, 22.4% xylan, 4.2% arabinan, 0.6%
mannan, 1.4% galactan, 3.8% uronyl, 11% lignin and 5.6% acetyl content.
4.2.2

Crude enzyme mixtures

Spezyme CP and Multifect Xylanase were a gift from Genencor (Danisco US Inc.,
Genencor Division, Rochester, NY), while Novo 188 (Sigma–Aldrich Corp., St. Louis, MO,
®

Novozyme 188 , C6105) was procured from Sigma. The protein concentration was determined
colorimetrically using the Pierce (Pierce Biotechnology, Rockford, USA) BCA (bicinchoninic
acid) assay kit with bovine serum albumin (BSA) as the standard [178].

45

4.2.3

Heterologous enzyme expression

Detail information is described in 3.2.
4.2.4

Protein purification

Detail information is described in 3.3.
4.2.5

Proteomics analysis

A brief overview to the proteomics methodology is presented here while the detailed protocol is
provided elsewhere [179]. Purified proteins of interest were denatured and reduced by adding
urea and thiourea to a final concentration of 7 M and 2 M, respectively. Fresh dithiothreitol was
added to a final concentration of 5mM, and samples were incubated at 60 °C for 30 min.
Following incubation, the protein sample was diluted 10-fold with 100 mM NH4HCO3, pH 8.4,
to reduce salt concentration. A volume of 1 M CaCl2 was added to the diluted sample to a final
concentration of 1 mM, and the sample was digested at 37 °C using sequencing grade trypsin
(Promega, Madison,WI) at a ratio of 1 unit/50 units of protein (1 unit =< 1 µg of protein) for 4 h.
Following incubation, digested samples were desalted using an appropriately sized C-18 SPE
column (Supelco, St. Louis, MO) and vacuum manifold. Three column volumes of methanol
were passed through the column followed by 2 column volumes of nanopure water. After
passing the sample through the column, the column was washed with 4 volumes of a 95%
acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) solution. Peptides were eluted using one
column volume of an 80% ACN, 0.1% TFA solution. The collected peptides were concentrated
to a final volume of 150 µl and measured using the BCA assay (Pierce Biotechnology, Rockfort,
IL) according to the manufacturer's instructions. Peptides from enzyme tryptic digests were
analyzed using high resolution reversed-phase HPLC separation coupled to an ion trap mass

46

spectrometer (LTQ, Thermo Electron, San Jose CA) that was operated in a data-dependent
MS/MS mode [179]. MS/MS spectra were analyzed using the SEQUEST algorithm in
conjunction with a protein collection of all fungal entries from Uniprot (Swiss-Prot and
TrEMBL). Preliminary filtering of identified peptides was performed using: a minimum crosscorrelation cut-off (XCorr) of either 1.9, 3.0, or 3.2 for 1+, 2+, or 3+ charge states, respectively;
partially and fully tryptic peptides (peptides that contained either an arginine or lysine at the site
of cleavage); DelCn >= 0.1. Estimation of peptide False Discovery Rate (FDR) was calculated
by decoy database searching techniques, resulting in values between 0.6% and 1.9%.
4.2.6

Enzyme activity assays

The enzyme activity assays were based on a high-throughput microplate based method as
described in previous work [177]. A 2.2 ml deep-well microplate (Lot # 780271, Greiner,
Monroe, NC) was used to add 250 µl of 1% (w/v) stock substrate (CMC, Avicel, oat spelt xylan,
cellobiose, xylobiose), 50 µl of 0.5 M citrate buffer (pH 5.0) and 200 µl of appropriately diluted
enzyme samples (20 ng to 100 µg/well). Carboxymethyl cellulose (CMC, Lot # 419273),
cellobiose (Lot # C7252), oat spelt xylan (Lot # 9559) and Avicel (Lot # 11365) were procured
o

from Sigma (Sigma-Aldrich, St. Louis, MO). The microplates were incubated at 50 C with
shaking at 200 rpm for 10 min (cellobiose), 60 min (CMC, xylan) or 300 min (Avicel). The
amount of glucose released was estimated using an enzyme assay kit (R-Biopharm, Marshall,
MI). One unit of cellobiase was defined as one micromole of glucose released per milligram
enzyme per minute under the assay conditions. For CMC, Avicel and xylan based substrates the
reducing sugars released were estimated using 3,5-dinitrosalicylic acid (DNS) based assay [180].
The hydrolyzate supernatants were filtered through 0.45 µm microplate filter (Lot # R6PN00144,
Millipore, Ireland) and 50 µl of the supernatant was incubated with 100 µl of DNS reagent in

47

polypropylene microplate wells (Lot # 651201, Greiner, NC) at 100 °C for 30 min. After the
plates cooled down to room temperature, 100 µl of the solution was transferred to a clear, flatbottom microplate (Lot # 353072, Becton Dickinson Labware, NJ, USA) for measuring
absorbance at 540 nm. Suitable reducing sugar standards (either glucose or xylose standards
ranging from 0.1-2 g/l) were included for the DNS assay. One unit of CMCase, Avicelase and
xylanase activity was defined as one micromole of reducing sugars (as glucose equivalents for
Avicel/CMC and xylose equivalents for xylan) released per milligram enzyme per minute under
the respective assay conditions.
The para-nitrophenyl (pNP) based chromogenic substrates used were 4-nitrophenyl-β-Dcellobioside (pNPC Lot # N5759), 4-nitrophenyl-β-D-glucopyranoside (pNPG Lot # N7006), 4nitrophenyl-β-D-xylopyranoside (pNPX Lot # N2132) and 4-nitrophenyl-α-L-arabinofuranoside
(pNPAf Lot # N3641)). All substrates were procured from Sigma (Sigma-Aldrich, St. Louis,
MO). The assay mixtures containing 80 µl of 1 mM pNP substrate, 10 µl of 0.5 M citrate buffer
(pH 5.0) and 10 µl of diluted enzymes (20 ng to 16 ug/well) in 350 µl micro plates were
o

incubated at 50 C with shaking at 200 rpm. After 15 min reaction time, 200 µl of 1M Na2CO3
was added to assay mixtures to arrest the hydrolytic reaction. The amount of pNP released was
quantified by measuring absorbance (at 420 nm) of 4-nitrophenol (pNP, Lot # 1048) based
standard curve (0.1 to 1 mM). One unit of enzyme activity was defined as one micromole of pnitrophenol released per milligram enzyme per minute under the assay conditions.
4.2.7

Enzymatic hydrolysis of AFEX treated corn stover

All six core enzymes were used to hydrolyze AFEX treated corn stover in various relative
amounts and total protein loadings to determine optimal enzyme combinations that maximize
glucan and xylan digestibility. Minitab (Version 15.0, Minitab Inc, State College, PA) statistical
48

software was used to create a suitable mixture optimization design and analyze responses. In a
mixture problem with q factors, it is common to define proportion variables xi, for i = 1, 2, ..., q,
where xi ≥ 0 represents the proportion of ingredient i in the mixture and x1+x2+…+xi +…+xq =1.
The proportion variable allows one to consider a particular mixture experiment as a geometric
point. In particular, the set of all points (x1, x2, ..., xq) whose coordinates satisfy xi ≥ 0 and
x1+x2+…+xi +…+xq =1 is called a q-dimensional simplex [181]. In this work, a fivecomponents
simplex centroid mixture design was generated for CBH I, CBH II, EG I, EX and βX which were
loaded at three different total protein loadings (7.5, 15 and 30 mg/g glucan). βG was loaded at 10%
of the total enzyme loading (0.75, 1.5 and 3 mg/g glucan, respectively) to simplify dimensions of
the experiment and this amount was later re-optimized. The hydrolysis data were analyzed by the
software to generate a mixture regression statistical model and used to predict optimum mixture
composition. The hydrolysis experiments were performed in 2.2 ml deep well microplates at 0.2%
(w/w) total glucan loading along with 50 mM citrate buffer (pH 5.0) in a total volume of 500 µl
o

per well [177]. The microplates were incubated at 50 C with shaking at 200 rpm for 24 h. All
experiments were carried out in quadruplicate; with mean values and standard deviations
reported.
4.2.8

Glucose and xylose assays

Glucose and xylose released after hydrolysis were assayed using enzymatic kits purchased from
R-Biopharm (Lot # 10716251035, Marshall, MI) and Megazyme (Lot # 80110-2, Bray, Ireland),
respectively. The glucose assay was based on a two step enzymatic reactions, where D-Glucose
was first phosphorylated to D-glucose-6-phosphate using ATP and hexokinase. The D-glucose-

49

+

6-phosphate was then reacted with NADP by glucose-6-phosphate dehydrogenase to form Dgluconate-6-phosphate and NADPH. The reactions are stoichiometric to the amount of Dglucose and the corresponding increase in NADPH was measured at 340 nm to estimate glucose
concentration. The xylose assay was based on an analogous two-step reaction method. α-Dxylose was first converted to isomeric β-D-xylose by xylose mutarotase. β-D-xylose was then
+

reacted with NAD to form D-xylonic acid and NADH. The corresponding increase of NADH
was measured at 340 nm to estimate xylose concentration.
4.3 Results and Discussion
4.3.1

Activity of purified enzymes

Enzyme sources (in order to assign respective glycosyl hydrolase family to each purified protein)
were determined through proteomics that helped identify the major tryptic peptides obtained for
all six purified proteins (two major tryptic peptides for each enzyme is listed in Table 4.1.

50

Table 4.1 Identification of major peptides from six purified glycosyl hydrolases by LC-MS/MS.
Observation counts on
MaxXc MaxDe
Peptides based on
Similari Uniprot
Enzyme
LC-MS/MS
orr
lCn
SEQUEST analysis
ty
KB No.
Total
Individual
Amino acid BLAST
Sequence
peptide
peptide
position
analysis
Purified
K.KLTVVTQFETSGAIN
18 AA
1602
475
6.6225 0.6159
100%
P62694
CBH I
R.Y
(303 - 320)
14 AA
400
4.0915 0.5665
K.YGTGYCDSQCPR.D
100%
P62694
(183 - 196)
Purified
11 AA
428
63
3.6351 0.3634
K.YKNYIDTIR.Q
100%
P07987
CBH II
(218 - 228)
R.TLLVIEPDSLANLVT
23 AA
48
7.5152 0.5898
100%
P07987
NLGTPK.C
(237 - 259)
Purified
R.LYLLDSDGEYVMLK.
16 AA
166
21
6.4641
0.444
100%
P07981
EG I
L
(130 - 145)
K.TFTIITQFNTDNGSPS
26 AA
18
6.0823 0.5831
100%
P07981
GNLVSITR.K
(269 - 294)
Purified
13 AA
Q30BH
2647
126
5.0463 0.5181
K.HYIAYEQEHFR.Q
100%
bG
(189 - 201)
9
R.DLANWNVETQDWEI
21 AA
Q30BH
126
6.9095 0.5488
100%
TSYPK.M
(820 - 840)
9
Purified
K.LGEVTSDGSVYDIYR
17 AA
397
121
5.5168 0.5299
100%
P36217
EX
.T
(136 - 152)
R.NPLIEYYIVENFGTY
25 AA
48
7.8233 0.5948
100%
P36217
NPSTGATK.L
(113 - 137)
Purified
R.SVMCSYNAVNGVPS
20 AA
2811
330
6.3165 0.4447
100% O42810
bX
CANK.F
(266 - 285)
R.SVVVKFELKGEEAVI
38 AA
272
7.7185 0.4754 LSWPEDTTSDFVSSIDG
100% O42810
(760 - 797)
GLDR.K

51

Organism

Trichoder
ma reesei
Trichoder
ma reesei
Trichoder
ma reesei
Trichoder
ma reesei
Trichoder
ma reesei
Trichoder
ma reesei
Aspergillu
s niger
Aspergillu
s niger
Trichoder
ma reesei
Trichoder
ma reesei
Aspergillu
s nidulans
Aspergillu
s nidulans

The purified enzymes were evaluated for their hydrolytic activity on various substrates to
determine activity and purity of isolated proteins. The activity of all enzymes with typical
polymeric substrates (i.e. Avicel, oat spelt xylan, CMC) and pNP based chromogenic substrates
was assessed. The activity assay results for all purified enzymes are shown in Table 4.1. The
endo-acting enzyme EG I had high specific activity on CMC (6.69 U) and xylan (5.08 U);
comparable to EX activity of xylan (8.24 U) as well. Interestingly, endoxylanase (and xylooligomerase) activity for EG I has been reported previously [182], suggesting that this enzyme
couple play a dual role in hydrolyzing glucan and xylan in pretreated cell walls, unlike other
endoglucanases. In addition, EG I showed significant activity on pNPC based chromogenic
substrate.
Table 4.2 Purified enzymes activity assay on chromogenic p-nitrophenyl (pNP) glycosidic
substrates, Avicel, CMC, oat spelt xylan, cellobiose and xylobiose.
Specific activity
pNPC
pNPG
pNPX
pNPAf

CBH I
0.0014
n.d.
n.d.
n.d.

CBH II
n.d.
n.d.
n.d.
n.d.

EG I
0.0466
n.d.
n.d.
n.d.

βG
2.47
4.15
0.00952
n.d.

EX
n.d.
n.d.
n.d.
n.d.

βX
n.d.
0.0063
1.272
0.142

Avicel

0.019

0.027

0.011

n.d.

n.d.

n.d.

Xylan

n.d.

n.d.

5.08

n.d.

8.24

n.d.

CMC

n.d.

n.d.

6.69

n.d.

n.d.

n.d.

Cellobiose

n.d.

n.d.

n.d.

124.9

n.d.

n.d.

Xylobiose

n.d.

n.d.

n.d.

n.d.

n.d.

52.2

One unit of specific activity was defined as one µmol pNP released per mg protein per
minute. One unit of specific activity was defined as one µmol (as glucose equivalents)
reducing sugars released based on DNS method (for Avicel and CMC) per mg protein per
min. For oat spelt xylan, specific activity was defined based on xylose equivalents. For
cellobiose and xylobiose, one unit of specific activity was denoted as one µmol of glucose or
xylose released per mg protein per minute. Where; n.d. is not detectable.

52

Among the exo-acting enzymes, CBH I and CBH II had significant activity on Avicel (0.019 and
0.027 U, respectively), compared to the minor activity of EG I (0.011 U) seen on Avicel as well.
This is not surprising considering Avicel has significant proportion of amorphous cellulose
(nearly 20-30%) [183]. Also, if the CBH’s are not extensively polished, they show significant
CMC activity (1.3 U and 1.6 U for CBH I and CBH II, respectively). Hence, the polishing steps
are crucial to remove endoglucanase contamination in cellobiohydrolases to obtain highly pure
enzymes. Purified CBH I showed much lower pNPC activity (0.0014 U) than EG I, while CBH
II had no detectable activity. βG and βX did not show appreciable activity on any of the
polysaccharide based substrates. βG and βX had significant activity on cellobiose (124.9 U) and
xylobiose (52.2 U), respectively. βG and βX also showed significant activity on pNPG (4.15 U)
and pNPX (1.27 U), respectively. In addition, βG also had significant activity on pNPC (2.47 U)
and trace activity on pNPX (0.0095 U). Similarly, βX has trace activity on pNPG (0.0063 U), but
no detectable activity on cellobiose, while βX activity on pNPAf (0.142 U) would indicate αarabinofuranosidase cross-activity. Similar cross-activity has been reported earlier for certain GH
3 β-xylosidases [184]. Other enzymes such as CBH II and EX had no detectable activities on any
of the chromogenic pNP substrates.
4.3.2

Hydrolysis of AFEX treated corn stover by purified enzymes

Core enzyme mixtures were tested for their hydrolysis performance on AFEX treated corn stover.
All enzyme loadings were based on equivalent bovine serum albumin BCA based measurement.
To simplify the matrix of enzyme combinations to be tested, βG was loaded at 10% of the total
cellulase/hemicellulase (CBH I + CBH II + EG + EX + βX) to prevent cellobiose inhibition. The
optimum βG loading was later determined for the optimized five enzyme mixture.

53

From Table 4.3 (Experiments #1-32), the reproducibility of the hydrolysis experiment is
satisfactory with low standard deviations (mostly < 2%) observed among quadruplicates. The
trends for both glucose and xylose yields among different enzyme mixtures was dependent on
the unique enzyme combinations. In terms of maximizing both glucose and xylose yield, the best
experimental mixture tested contained CBH I, CBH II, EG I, EX and βX at equal protein loading
(#31). Compared to Spezyme CP (at equal protein loading), glucose yield was 10-50% higher,
whereas, xylan conversion was 40-225% higher, depending on total protein loading employed.
Replacing the cellulase fraction with suitable hemicellulases (#29) helps increase xylan
conversion by 500-1000% (with respect to #7) without causing significant change in glucan
conversion. Selig et al. [185] reported 80-150% increase in xylan conversion to xylobiose due to
supplementation of CBH I with an endoxylanase (from Thermomyces lanuginosus), with a
corresponding increase of 15-20% in the glucan conversion to cellobiose during hydrolysis of
hot water treated corn stover. However, in that experiment the protein loading for the
endoxylanase supplementation was 5-15% higher, hence explaining the slight improvement in
the overall glucan conversion as well. Individual enzymes (along with βG) generated very
limited amount of glucose and xylose (#1 to #5). Interestingly, significant xylan conversion was
obtained by only EG I (#3), which is not unexpected based on its significant oat spelt xylan
activity. It may be possible that bi-functional enzymes like EG I have an advantage over
endoglucanases from other GH families (e.g. GH 5, GH 12, GH 61) due to the fact that cellulose
microfibrils are thought to be enclosed within hemicellulose rich sheaths [186] that would hinder
the activity of mono-functional endoglucanases.
For binary enzyme combinations (#6 to #15), CBH I/EG I mixture (#7) gave the highest glucose
yield (62.6% at 33 mg/g glucan) and the EX/βX mixture (#15) gave the highest xylose yield

54

(51.8% at 33 mg/g glucan) followed by EG I/βX (#14, 40.5% at 33 mg/g glucan) likely due to
EG I’s xylan cross-activity. Interestingly, it was possible to achieve close to 50% xylan
conversion in the absence of any cellulases (< 5 % glucan conversion, #15). This suggests that
the cell wall ultra-structure was significantly modified during AFEX pretreatment that allowed
significant enzymatic accessibility to both glucan and xylan fractions, unlike untreated cell walls
(data not shown) [187]. Interestingly, even though CBH II had significantly higher activity (42%
higher) on crystalline cellulose than CBH I (Table 4.3), CBH I gave significantly higher
conversions (45-50% higher) in binary/ternary/quartenary mixtures that had either one of the
CBH’s (Compare #7 vs. 10; 19 vs. 22; 29 vs. 30). This result reiterates the importance of
optimizing enzyme cocktails on real pretreated lignocellulosic biomass and not on artificial
cellulosic substrates like Avicel [114]
For ternary enzyme combinations (#16 to #25), CBH I/CBH II/EG I mixture (#16) gave the
highest glucose yield but very low xylose yield. Binary mixtures of CBH I + EG I synergized
together to give higher combined glucan and xylan conversion (1.5–2.5 fold, #7) compared to
CBH I + EX, CBH II + EG I and CBH II + EX. However, for ternary systems that included CBH
II the combined glucan and xylan conversions for CBH I + CBH II with either EG I or EX are
remarkably similar. This suggests that presence of CBH II reduces the advantage of EG I over
EX when synergizing with CBH I alone, possibly due to minor endo-activity typically seen for
CBH II [156] In order to get significantly higher xylose yield, either the EG I-βX or EX-βX
combination is necessary. For quarternary enzyme combinations (#26 to #30), without βX, xylan
conversion dropped dramatically (#26) and without CBH I, glucan conversion decreased as well
(#30).

55

From Table 4.3, it is not unexpected to find that CBH I, CBH II and EG I are critical for glucan
hydrolysis; while EG I, EX and βX are important for xylan hydrolysis. By plotting glucose yield
versus xylose yield at varying total enzyme loading (Figure 4.1), the glucose yield was found to
be scattered from almost 0% to 75% while xylan conversion clustered into two sets. As long as
EX or EG I and βX are present within the mixture (cluster 1), xylan conversion was found to be
greater than 30% compared to cluster 2. Also, EX/βX mixture has higher xylose yield compared
with EG I/βX containing mixtures. By comparing hydrolysis yields among different protein
loadings, an interesting result comes to light. Reducing enzyme loading by 4 fold from 33 to 8.25
mg/g glucan resulted in quite different effects on overall glucose and xylose hydrolysis yields.
For enzyme mixtures giving either higher or lower glucose yield, reduction of total enzyme
loading resulted in a consistent drop of around 50% in glucose yield with no apparent correlation
to initial glucose yield (33 mg/g glucan loading). However, xylan conversions dropped
depending on the type of enzyme mixture. Enzyme mixtures (inclusive of EX/EG + βX, Cluster
1) that contributed to higher xylose yields saw a lower decrease in xylan conversions upon
reducing enzyme loading by four-fold, compared to the low xylan hydrolyzing enzyme mixtures
(Cluster 2). This suggests that it might be possible to further reduce hemicellulase loading
without sacrificing xylan hydrolysis yields. Increasing enzyme loading helps increase glucose
and xylose hydrolysis yields. However, the extent of improvement is quite different. Glucose
yield increases by more than 20% while xylan conversion increases by less than 10% upon
doubling the enzyme loading. The highest xylose yield was always below 60% conversion. This
might be due to lack of other accessory enzyme activities such as α-arabinofuranosidase,
feruloyl/acetyl xylan esterases, pectinases and α-glucuronidase.

56

Xylan conversion

33 mg/g glucan
60%

16.5 mg/g glucan

8.25 mg/g glucan

40%

20%

0%
0%

20%

40%

60%

80%

Glucan conversion
Figure 4.1 Twenty four hours glucose versus xylose yields for different total enzyme
loading (33, 16.5 and 8.25 mg/g glucan) saccharifying AFEX treated corn stover. Abov the
Above
bar, all enzyme mixtures contain either EG I/
I/βX or EX/βX (Cluster 1) whereas below the
X
bar (Cluster 2) no such combinations exist.

57

Table 4.3 24 hours hydrolysis yields of AFEX treated corn stover by various enzyme
mixtures at three different total protein loadings. βG was loaded at 10% (mass loading) of
all other proteins. An equivalent amount of Spezyme (equivalent mass basis 33, 16.5 and
8.25 mg/g glucan, respectively; with no βG supplementation) was included.
Enzymes ratio
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32

CBH I CBH II
1
0
0
1
0
0
0
0
0
0
0.5
0.5
0.5
0
0.5
0
0.5
0
0
0.5
0
0.5
0
0.5
0
0
0
0
0
0
0.33
0.33
0.33
0.33
0.33
0.33
0.33
0
0.33
0
0.33
0
0
0.33
0
0.33
0
0.33
0
0
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0
0
0.25
0.2
0.2
Spezyme CP

EG I
0
0
1
0
0
0
0.5
0
0
0.5
0
0
0.5
0.5
0
0.33
0
0
0.33
0.33
0
0.33
0.33
0
0.33
0.25
0.25
0
0.25
0.25
0.2

EX
0
0
0
1
0
0
0
0.5
0
0
0.5
0
0.5
0
0.5
0
0.33
0
0.33
0
0.33
0.33
0
0.33
0.33
0.25
0
0.25
0.25
0.25
0.2

βX
0
0
0
0
1
0
0
0
0.5
0
0
0.5
0
0.5
0.5
0
0
0.33
0
0.33
0.33
0
0.33
0.33
0.33
0
0.25
0.25
0.25
0.25
0.2

58

30 mg/g glucan plus 10% βG
Glucose yield Xylose yield
Avg
Stdev Avg
Stdev
11.5% 1.0% 4.1% 0.5%
7.6% 0.2% 0.0% 1.6%
16.2% 0.8% 11.3% 2.4%
3.1% 0.5% 22.4% 2.0%
2.4% 0.4% 8.5% 2.3%
15.9% 0.8% 2.3% 0.5%
62.6% 0.9% 9.4% 0.8%
29.3% 1.3% 19.2% 0.7%
10.0% 1.4% 12.5% 0.8%
41.1% 0.8% 9.0% 1.4%
24.5% 1.1% 16.9% 1.4%
7.0% 0.5% 6.8% 1.5%
15.7% 0.9% 17.5% 2.0%
14.7% 1.2% 40.5% 1.8%
2.6% 1.1% 51.8% 2.6%
68.5% 1.7% 9.2% 0.5%
58.3% 1.7% 13.4% 1.2%
15.7% 0.7% 11.5% 1.2%
64.9% 1.8% 13.2% 1.4%
57.9% 1.1% 45.8% 1.0%
28.1% 2.2% 49.8% 2.3%
44.3% 0.8% 13.8% 1.2%
38.8% 1.4% 42.0% 0.5%
21.2% 0.8% 51.1% 1.5%
13.9% 0.6% 50.3% 1.4%
73.7% 1.4% 12.2% 1.0%
66.3% 1.6% 43.5% 1.0%
53.2% 0.9% 50.8% 0.6%
64.0% 1.5% 56.6% 2.9%
43.5% 0.5% 53.3% 1.6%
74.2% 1.5% 55.5% 1.6%
66.8% 1.3% 39.1% 1.5%

Table 4.3 (cont’d)

Enzymes ratio
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32

CBH I CBH II
1
0
0
1
0
0
0
0
0
0
0.5
0.5
0.5
0
0.5
0
0.5
0
0
0.5
0
0.5
0
0.5
0
0
0
0
0
0
0.33
0.33
0.33
0.33
0.33
0.33
0.33
0
0.33
0
0.33
0
0
0.33
0
0.33
0
0.33
0
0
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0
0
0.25
0.2
0.2
Spezyme CP

EG I
0
0
1
0
0
0
0.5
0
0
0.5
0
0
0.5
0.5
0
0.33
0
0
0.33
0.33
0
0.33
0.33
0
0.33
0.25
0.25
0
0.25
0.25
0.2

EX
0
0
0
1
0
0
0
0.5
0
0
0.5
0
0.5
0
0.5
0
0.33
0
0.33
0
0.33
0.33
0
0.33
0.33
0.25
0
0.25
0.25
0.25
0.2

βX
0
0
0
0
1
0
0
0
0.5
0
0
0.5
0
0.5
0.5
0
0
0.33
0
0.33
0.33
0
0.33
0.33
0.33
0
0.25
0.25
0.25
0.25
0.2

59

15 mg/g glucan plus 10% βG
Glucose yield Xylose yield
Avg
Stdev Avg
Stdev
5.4% 0.7% 1.3% 1.0%
6.1% 1.0% 0.2% 1.0%
12.1% 0.5% 6.5% 1.7%
2.2% 0.2% 16.0% 0.9%
1.8% 0.2% 8.2% 0.3%
9.7% 0.9% 0.3% 0.7%
47.9% 0.9% 5.8% 0.6%
11.6% 1.0% 13.5% 1.0%
5.1% 0.2% 9.6% 0.6%
30.5% 1.1% 5.3% 0.6%
14.9% 1.0% 13.3% 0.7%
5.4% 0.4% 6.8% 0.7%
12.3% 0.7% 12.7% 0.8%
11.3% 0.7% 36.0% 1.5%
1.7% 1.1% 49.6% 1.3%
56.8% 2.2% 5.4% 0.7%
36.7% 1.6% 10.5% 1.2%
9.3% 0.7% 8.2% 0.3%
52.1% 1.0% 9.8% 1.3%
42.9% 0.5% 37.4% 0.7%
12.3% 2.5% 47.1% 1.1%
35.1% 0.7% 10.7% 0.8%
28.0% 1.1% 35.5% 1.9%
12.5% 0.9% 47.9% 1.9%
11.8% 0.6% 47.5% 0.3%
65.3% 0.8% 9.1% 1.1%
53.7% 1.2% 37.8% 1.4%
30.5% 1.3% 48.1% 1.0%
48.4% 1.2% 50.5% 0.9%
31.2% 0.9% 47.8% 0.4%
64.1% 1.4% 51.4% 1.6%
47.3% 1.4% 22.9% 2.8%

Table 4.3 (cont’d)

Enzymes ratio
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32

CBH I CBH II
1
0
0
1
0
0
0
0
0
0
0.5
0.5
0.5
0
0.5
0
0.5
0
0
0.5
0
0.5
0
0.5
0
0
0
0
0
0
0.33
0.33
0.33
0.33
0.33
0.33
0.33
0
0.33
0
0.33
0
0
0.33
0
0.33
0
0.33
0
0
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0
0
0.25
0.2
0.2
Spezyme CP

EG I
0
0
1
0
0
0
0.5
0
0
0.5
0
0
0.5
0.5
0
0.33
0
0
0.33
0.33
0
0.33
0.33
0
0.33
0.25
0.25
0
0.25
0.25
0.2

EX
0
0
0
1
0
0
0
0.5
0
0
0.5
0
0.5
0
0.5
0
0.33
0
0.33
0
0.33
0.33
0
0.33
0.33
0.25
0
0.25
0.25
0.25
0.2

βX
0
0
0
0
1
0
0
0
0.5
0
0
0.5
0
0.5
0.5
0
0
0.33
0
0.33
0.33
0
0.33
0.33
0.33
0
0.25
0.25
0.25
0.25
0.2

60

7.5 mg/g glucan plus 10% βG
Glucose yield Xylose yield
Avg
Stdev Avg
Stdev
5.5% 3.1% 0.7% 0.3%
4.6% 0.5% 0.8% 0.6%
9.7% 0.5% 4.5% 0.5%
1.1% 0.8% 10.9% 0.3%
1.1% 0.9% 6.6% 0.9%
5.9% 0.2% 0.7% 0.3%
32.8% 1.6% 4.1% 0.5%
5.2% 1.2% 8.8% 1.9%
3.0% 0.4% 7.3% 0.9%
23.1% 1.2% 4.1% 0.7%
8.1% 0.5% 8.7% 0.5%
3.8% 0.4% 5.6% 0.6%
8.8% 0.9% 8.8% 0.2%
8.0% 0.3% 30.6% 0.6%
0.6% 0.2% 44.5% 1.0%
42.0% 1.1% 3.0% 0.5%
16.7% 1.6% 7.6% 1.0%
6.4% 1.3% 5.1% 1.9%
37.3% 1.4% 7.6% 0.7%
29.7% 0.6% 31.0% 1.4%
5.9% 1.1% 42.4% 1.2%
25.6% 0.4% 7.8% 0.5%
19.7% 0.3% 32.1% 3.4%
8.3% 0.8% 43.3% 0.8%
10.3% 1.1% 43.4% 1.2%
51.5% 1.3% 6.4% 0.7%
39.9% 1.1% 31.4% 0.9%
13.6% 0.4% 40.4% 0.8%
33.3% 0.4% 45.0% 0.9%
22.7% 0.5% 42.5% 1.5%
47.0% 0.7% 44.4% 1.1%
30.6% 0.8% 13.7% 1.7%

4.3.3

Optimal core enzyme mixture for hydrolyzing AFEX treated corn stover

Table 4.4 Statistical mixture model regression analysis for glucose hydrolysis yields at three
total protein loadings for saccharifying AFEX treated corn stover.
30 mg/g glucan
Coefficient P value
Terms
*
CBH I
0.2231
*
CBH II
0.0923
*
EG I
0.1699
*
EX
0.1192
*
βX
0.0573
<0.001
CBH I*EG I
1.684
<0.001
CBH II*EG I
1.1135
<0.001
CBH II*EX
0.6724
<0.001
CBH I*CBH II*EG I
5.246
<0.001
CBH I*CBH II*EX
9.3207
<0.001
CBH I*EG I*EX
8.5795
<0.001
CBH I*EG I*βX
7.681
<0.001
CBH II*EG I*EX
3.3146
<0.001
CBH II*EG I*βX
4.8307
0.002
EG I*EX*βX
2.4303
0.023
CBH I*CBH II*EG I*βX 20.7769
<0.001
CBH I*CBH II*EX*βX 58.0654
2
96.48%
Regression R
2

R (predicted)

7.5 mg/g glucan
Coefficient P value
0.0611
*
0.0516
*
0.1065
*
0.0203
*
0.0221
*
<0.001
0.9213
<0.001
0.5656
0.1958
0.001
<0.001
5.4967
<0.001
3.3075
<0.001
6.7795
<0.001
3.8956
<0.001
3.9445
<0.001
2.1095
<0.001
2.1053
<0.001
23.8943
<0.001
15.5443
97.15%

96.87%

93.48%

15 mg/g glucan
Coefficient P value
*
0.0965
*
0.0737
*
0.1345
*
0.055
*
0.037
<0.001
1.4036
<0.001
0.774
<0.001
0.3758
<0.001
6.1749
<0.001
6.8796
<0.001
8.3711
<0.001
5.7115
<0.001
4.2069
<0.001
3.2345
<0.001
2.3416
<0.001
27.1232
<0.001
34.6422
97.57%

96.15%

Cellulose comprises the largest polysaccharide fraction in lignocellulosic cell walls and glucose
is the preferred carbon substrate for many microorganisms. In order to further enhance the
glucose yield, the enzyme mixture composition and glucan conversion data (Table 4.3) were
analyzed by Minitab to develop a statistically based predictive model. Response surface models
were generated for all three total protein loading experiments. Model terms, coefficients and P
values are listed in Table 4.4. Only terms that have a significantly low P value (<0.05) have been
included to develop statistically valid and highly predictive models. The model explains the

61

2

variance in the data very well with R greater than 0.9. The optimum mixture composition
(capable of giving the highest glucan conversion) from the response surface models was
predicted using the response optimizer functionality available in Minitab. The optimized mixture
composition and predicted responses are shown in Table 4.5 and Figure 4.4.
The coefficients of the model also provide insight into ranking the importance of each enzyme
and synergistic interactions among various enzymes on the hydrolysis yields. The model clearly
indicates that both EX and βX work synergistically with cellulases and can help significantly
increase glucose yields. This might be due to the removal of hemicelluloses wrapped around
glucan microfibrils to help increase cellulose accessibility [33] and also prevention of cellulase
inhibition by hemicellulose oligomers [188]. Another interesting trend regarding relative
importance of EG I based on total protein loading was noticed from the predicted coefficients of
the model. This trend was harder to notice by simply glancing over the original dataset in Table
4.4. Ranking coefficients for single, binary, ternary and quaternary terms consistently
highlighted EG I containing terms at the top of each list for the lowest total protein loading
(0.1065 for EG I, 0.9213 for CBH I*EG I, 6.7795 for CBH I*EG I*EX and 23.8943 for CBH
I*CBH II*EG I*βX). However, for the 30 mg/g glucan enzyme loading model most terms with
the highest coefficient within each of the respective term families (i.e. single, ternary, quaternary)
do not include EG I (0.2231 for CBH I, 9.3207 for CBH I*CBH II*EX and 58.0654 for CBH
I*CBH II*EX*βX). This statistical result may have a phenomenological explanation based on
substrate inhibition encountered at lower enzyme to substrate ratios [189]. At lower enzyme
loadings, the CBH enzymes bound to the cellulosic substrate are further separated from potential
reducing ends than at higher protein loadings (for identical relative enzyme ratios) due to relative
surface density of the enzymes on the substrate. Therefore, in order to maximize glucan

62

digestibility at lower total protein loadings it would be necessary to increase the number of
accessible reducing ends for CBH enzymes, possibly through increasing the relative EG I
loading. This may help explain why the model predicts 15% higher EG I ratio (35% vs. 30%) in
the lowest total protein loading compared to the 30 mg/g glucan enzyme loading optimum
mixture (Table 4.5). However, in order to validate this hypothesis suitable adsorption
experiments using purified cellulases on AFEX corn stover would need to be conducted at
varying total protein loadings. The optimum mixtures for all three total enzyme loadings are
similar, containing approximately 29-30% CBH I, 18-20% CBH II, 30-35% EG I, 14-15% EX
and 2-6% βX based on total protein mass loading (excluding βG). These results also validate
earlier findings that hemicellulase supplementation can help reduce cellulase loading while
giving higher overall hydrolysis yields [185, 190].
The ternary plots (Figure 4.2) were generated by the regression model given in Table 4.4. The
contour patterns of different protein loadings were quite congruous indicating that the optimum
enzyme ratios were overlapping, though the three-dimensional shapes are quite different (due to
variable slopes between low and high protein loading). By fixing EX and βX at their optimum
ratio, the effect of varying CBH I, CBH II and EG I absolute ratios on glucose yield was
determined. All three enzymes were found to be important. By increasing the total enzyme
loading (A.1 to A.3), the glucose yield became less sensitive to relative ratios of CBH I, CBH II
and EG I. Similar results are also seen in (B.1 to B.3). EX can increase hydrolysis yields and a
small amount of βX (around 4%) was critical. Since xylan conversions are highly dependent on
βX, including βX in the mixture was essential. The contour plots help visually demonstrate that
the relative ratio of enzymes becomes less important at relatively higher total protein loadings, as
long as there is a minimal amount of each individual component. This is an important finding, as

63

for a feasible cellulosic ethanol process, enzyme usage needs to be substantially minimized. This
is possible via gravitating towards significantly lower protein loadings than what are currently
utilized in most published research articles (e.g. reducing enzyme usage from 15 FPU/g glucan to
say 5 FPU/g glucan; where 15 FPU typically corresponds to 25-35 mg enzyme/g glucan). This
reduction in enzyme usage would be further facilitated by consumption of hydrolyzed sugars
during the co-fermentation step (i.e. SSF).

64

Figure 4.2 Ternary plots of models developed to predict glucose yields as a function of
varying enzyme loadings upon hydrolysis of AFEX treated corn stover. Expected glucose
yield ranges are denoted by the different colors/patterns in the legend. A.1, A.2 and A.3 are
different
ternary plots of CBH I, CBH II and EG I, with EX and βX held constant at 0.15 and 0.04,
X
0.0
respectively (as protein mass ratio where sum of all five protein ratios is unity). B.1, B.2
and B.3 are ternary plots of CBH I, EX and βX, with CBH II and EG I held constant at
ry
X,
0.19 and 0.32 ratio. A.1/B.1, A.2/B.2 and A.3/B.3 are plots of 7.5, 15 and 30 mg/g glucan
enzyme loading, respectively. For all experiments, βG was loaded at 10% of the total
G
enzyme (CBH I + CBH II + EG I + EX + βX) mass loading.

65

Figure 4.2 (cont’d)

In order to determine the minimal βG loading necessary, increasing amounts of βG were loaded
G
to the optimal cocktail (predicted from previous experiments) for a total five enzyme (CBH I +
CBH II + EG I + EX + βX) loading at 7.5, 15 and 30 mg/ g glucan. The results (shown in Figure
X)
4.3) indicate that 1-5% of βG (i.e. 0.375, 0.75, 1.5 mg/g glucan, respectively, for all three total
G
66

protein loadings) is more than sufficient to prevent end-product inhibition. Xylose yield was not
significantly affected by βG supplementation. The optimum mixtures for all three total enzyme
loadings, inclusive of all six core enzymes, would probably contain 27-30% CBH I, 17-20%
CBH II, 29-35% EG I, 14-15% EX, 2-6% βX and 1-5% βG based on total protein mass loading.
The statistical model predictions were tested for all three total protein loadings with βG
supplementation at 10% of CBH I + CBH II + EG I + EX + βX (shown in Table 4.5). Since all
optimally predicted combinations had around 15% EX and 4% βX loading, the xylan
conversions were comparable to the data obtained in earlier designed experiments (Table 4.3).
For 15 mg/g glucan loading, the model prediction closely matched the experimental data set. At
7.5 mg/g glucan, the result was 2% higher than the model expectation while at 30 mg/g glucan
the result was 2% lower than model expectation. Compared with the best experimental
combination (Table 4.3, #31), the glucose yield for optimized mixtures increased further (7.7%,
6.7% and 10.5% corresponding to 7.5, 15 and 30 mg/g glucan, respectively). Compared to
Spezyme CP, where individual enzyme ratios have not been optimized for AFEX treated corn
stover, the optimized cocktail gave much higher glucose and xylose yields. Also, when the
enzyme loading was reduced by 4 fold from 33 to 8.25 mg/g glucan, the drop in the glucose and
xylose yield was much lower for the optimized cocktail compared to Spezyme CP. For Spezyme
CP, the glucose yield decreased from 66.8% to 30.6% and xylose yield decreased from 39.1% to
13.7%, whereas the optimized cocktail showed a decrease from 80% to 52.6% and 56.2% to 42.2%
for glucose and xylose yields, respectively. This finding reconfirms the importance to tailormake enzyme cocktails for pertinent pretreated substrates in order to minimize enzyme usage,
maximize digestibility and hence reduce the overall cost of producing cellulosic ethanol.

67

90%
80%

Glucose yield

70%
60%
50%
30 mg/g glucan

40%

15 mg/g glucan
30%

7.5 mg/g glucan

20%
10%
0%
0% 0.1% 0.5% 1%

2%

5% 10% 20%

beta-glucosidase loading of the remaining enzyme mixture
70%
60%

Xylose yield

50%
40%
30 mg/g glucan
30%

15 mg/g glucan
7.5 mg/g glucan

20%
10%
0%
0% 0.1% 0.5% 1%

2%

5% 10% 20%

beta-glucosidase loading of the remaining enzyme mixture

Figure 4.3 Twenty-four hours glucose (A) and xylose (B) yields for saccharified AFEXtreated corn stover for varying bG loadings, held at fixed respective optimum ratios of
CBH I,CBH II, EG I, EX and bX for three different total enzyme loadings.

68

Table 4.5 24 hours glucose yield for AFEX treated corn stover saccharified at optimum enzyme loading. The model generated
optimum mixture hydrolysis predictions were tested at varying total enzyme loadings.
5 core
enzyme
loading
(mg/g
glucan)
30
15
7.5

βG
enzyme
loading
(mg/g
glucan)
3
1.5
0.75

5 core enzyme mixture ratios (mass
basis)
CBH
I

CBH
II

EG I

EX

βX

0.305
0.296
0.293

0.182
0.192
0.193

0.300
0.323
0.352

0.152
0.149
0.141

0.061
0.040
0.021

Glucose yield
Model
Expectatio
n
82.0%
68.4%
50.6%

69

Experime
ntal
Average
80.0%
67.4%
52.6%

Xylose yield
Standard
Deviation
1.3%
0.3%
0.2%

Experime
ntal
Average
56.2%
48.3%
42.2%

Standard
Deviation
1.2%
0.1%
0.4%

A

90%
80%

Glucose yield

70%

Spezyme CP
Best mixture in 31 combination
Tested optimized mixtures

60%
50%
40%
30%
20%
10%
0%
8.25 mg/g glucan

B 70%
60%

16.5 mg/g glucan

33 mg/g glucan

Spezyme CP
Best mixture in 31 combination
Tested optimized mixtures

Xylose yield

50%
40%
30%
20%
10%
0%
8.25 mg/g glucan

16.5 mg/g glucan

33 mg/g glucan

Figure 4.4 Glucose (A) and xylose (B) yield for different enzyme cocktails at 24 hours.

70

4.4 Conclusion
We have successfully isolated six core cellulases and hemicellulases through various purification
and heterologous expression strategies. Thirty one unique combinations of purified fungal
glycosyl hydrolase mixtures were tested on AFEX treated corn stover to determine glucose and
xylose yields, at three total protein loadings (8.25, 16.5 and 33 mg/g glucan; inclusive of all six
enzymes) using a suitable design of experiments methodology. The optimal enzyme ratios that
maximized hydrolysis yields were found to be strongly dependent on the total enzyme loading
employed, with endoglucanase I (EG I) playing a relatively more important role at lower total
protein loadings. Reducing relative cellulase ratio by addition of suitable hemicellulases
(endoxylanase, β-xylosidase) helped significantly enhance xylose yield with no decrease in
glucose yield. The range of optimal ratios for the cocktail containing six core enzymes,
maximizing glucan and xylan hydrolysis yields of AFEX treated corn stover, is comprised of 2730% CBH I, 17-20% CBH II, 29-35% EG I, 14-15% EX, 2-6% βX and 1-5% βG based on total
protein mass loading. These results demonstrate the potential to rationally design an enzyme
mixture targeted towards a particular feedstock and pretreatment that can help maximize
hydrolysis yields and minimize protein usage in a cellulosic biorefinery.

71

CHAPTER 5 STRATEGY FOR IDENTIFICATION OF NOVEL FUNGAL AND
BACTERIAL GLYCOSYL HYDROLASE HYBRID MIXTURES
5.1 Introduction
Previous chapter demonstrate how fungal enzymes work synergistically. However, in nature,
anaerobic bacterial enzymes are typically aggregated and assembled on a complex scaffold
structure through various integrating modules known as cohesins and dockerins [191]. These
enzyme complexes, known as cellulosomes, are attached to the surface of the bacterial cell walls
[192-194]. Few studies have investigated the synergism among catalytic domains of various
bacterial enzymes, and the synergistic interactions between bacterial and fungal hydrolases
acting on pretreated lignocellulosic biomass. Some reports have shown exo/exo and exo/endo
synergism between fungal and bacterial enzymes hydrolyzing crystalline cellulose [195, 196].
Recent publications have reported synergy between Trichoderma and Serratia/Streptomyces
based on chitin degrading hydrolases completely hydrolyzing untreated crab shells [197]. But,
very few reports are available on the nature of synergistic interactions between bacterial and
fungal enzymes, especially bacterial hemicellulases hydrolyzing pretreated lignocellulosic
biomass.
In this chapter, the enzymatic digestibility of Ammonia Fiber Expansion (AFEX) treated corn
stover was evaluated by varying combinations of fungal and bacterial glycosyl hydrolases.
Fungal enzymes (CBH I, CBH II and EG I) were purified from suitable commercial sources
(Spezyme CP); while βG was purified from Novozyme 188. Two cellulases (LC1 and LC2), two
xylanases (LX1 and LX2), one β-glucosidase (LβG) and one β-xylosidase (LβX) were obtained
from various bacterial sources (e.g. Clostridium, Geobacillus, Dictyoglomus). This paper

72

presents a rational four-step strategy for designing an optimal enzyme cocktail, based on
enzymes from multiple sources, to efficiently hydrolyze pretreated lignocellulosic biomass to
help ultimately decrease the cost of cellulosic ethanol.
5.2 Materials and Methods
5.2.1

AFEX pretreatment

Detailed information is described in 4.2.1
5.2.2

Discovery and cloning of LX1, LX2, LβX and LβG

Detailed information is published by Gao et al[198]. Those discovery and cloning work have
been done by Lucigen.
5.2.3

Discovery and cloning of LC1 and LC2

Detailed information is published by Gao et al[198]. The protein sequences and strain sources
are listed in Table 5.1.

73

Table 5.1 Amino acid sequences and glycosyl hydrolase families for all six bacterial enzymes

Notat
ion

LC1

LC2

LX 1

LX 2

Unipr
ot No.

Source, name
and
predicted
molecular
weight

5

B8DZ
M2

Dictyoglomus
turgidum
(Dtur_0670,
37 kDa)

5

P0C2S
3

Clostridium
thermocellum
(CELC, 40.9
kDa)

10

B4BM
E8

Geobacillus
sp. G11MC16
(47.4 kDa)

P4570
3

Geobacillus
stearothermop
hilus
(XynA, 38.5
kDa)

GH
fami
ly

Amino acid sequence

MNNLPIKRGINFGDALEAPYEGAWSGYIIKDEYFKIVKDAGFDHVRIPIKWSVYTQ
KEAPYSIEKRIFDRVDHLIEEGLKNNLHVIINIHHYEEIMEDPLGEKERFLAIWRQIS
EHYKDYPNNLYFELLNEPTQNLSSELWNQFLKEAIEVIRRTNPERKIIVGPDNWNS
LYNLEKLIIPENDENIIITFHYYNPFPFTHQGAGWVKIDLPVGVKWLGTEEEKREIER
ELDMAVSWAEEHGNIPLYMGEFGAYSKADMESRVRWTDFVARSAEKRGIAWSY
WEFYSGFGVFDPEKNEWRTPLLRALIPERNI*
MVSFKAGINLGGWISQYQVFSKEHFDTFITEKDIETIAEAGFDHVRLPFDYPIIESDD
NVGEYKEDGLSYIDRCLEWCKKYNLGLVLDMHHAPGYRFQDFKTSTLFEDPNQQ
KRFVDIWRFLAKRYINEREHIAFELLNEVVEPDSTRWNKLMLEYIKAIREIDSTMW
LYIGGNNYNSPDELKNLADIDDDYIVYNFHFYNPFFFTHQKAHWSESAMAYNRTV
KYPGQYEGIEEFVKNNPKYSFMMELNNLKLNKELLRKDLKPAIEFREKKKCKLYC
GEFGVIAIADLESRIKWHEDYISLLEEYDIGGAVWNYKKMDFEIYNEDRKPVSQEL
VNILARRKT*
MAKTEQSYAKKPQISALHAPQLDQRYKDSFTIGAAVEPYQLLNEKDAQMLKRHF
NSIVAENVMKPINIQPEEGKFNFAEADQIVRFAKKHHMDIRFHTLVWHSQVPQWF
FLDKEGQPMVNETDPVKREQNKQLLLKRIETHIKTIVERYKDDIKYWDVVNEVVG
DDGELRDSPWYQIAGIDYIKVAFQTARKYGGNKIKLYINDYNTEVEPKRSALYNL
VKQLKEEGIPIDGIGHQSHIQIDWPSEEEIEKTIIMFADLGLDNQITELDVSMYGWPP
RAYLSYDAIPEQKFLDQADRYDRLFKLYEKLSDKISNVTFWGIADNHTWLDSRAD
VYYDTDGNVIVDPKAPYTRVEKGNGKDAPFVFDPEYNVKPAYWAIIDHK*
MCSSIPSLREVFANDFRIGAAVNPVTLEAQQSLLIRHVNSLTAENHMKFEHLQPEE
GRFTFDIAIKSSTSPFSSHGVRGHTLVWHNQTPSWVFQDSQGHFVGRDVLLERMK
SHISTVVQRYKGKVYCWDVVNEAVADEGSEWLRSSTWRQIIGDDFIQQAFLYAHE
ADPEALLFYNDYNECFPEKREKIYTLVKSLRDKGIPIHGIGMQAHWSLTRPTLDEIR
AAIERYASLGVILHITELDISMFEFDDHRKDLAAPTNEMVERQAERYEQIFSLFKEY
RDVIQNVTFWGIADDHTWLDHFPVQGRKNWPLLFDEQHNPKPAFWRVVNI*

74

10

Table 5.1 (cont’d)

βX

βG

MPTNVFFNAHHSPVGAFASFTLGFPGKSGGLDLELARPPRQNVFIGVESSHEPGL
YHILPFAETAGEDESKRYDIENPDPNPQKPNILIPFAKERIEREFRVATDTWKAGD
LTLTIYSPVKAVPDPETASEEELKLALVPAVIVEMTIDNTNGTRTRRAFFGFEGTD
PYTSMRRIDDTCPQLRGVGQGRILGIASKDEGVRSALHFSMEDILTATLEENWTF
GLGKVGALIADVPAGEKKTYQFAVCFYRGGYVTAGMDASYFYTRFFHNIEEVG
LYALEQAEVLKEQAFCSNELIEKEWLSDDQKFMMAHAIRSYYGNTQLLEHEGK
PIWVVNEGEYRMMNTFDLTVDQLFFELKMNPWTVKNVLDFYVERYSYEDRVR
FPGDETEYPGGISFTHDMGVANTFSRPHYSSYELYGISGCFSHMTHEQLVNWVL
CAAVYIEQTKDWAWRDRRLTILEQCLESMVRRDHPDPEKRNGVMGLDSTRTM
GGAEITTYDSLDVSLGQARNNLYLAGKCWAAYVALEKLFRDVGKEELAALAGE
QAEKCAATIVSHVTEDGYIPAVMGEGNDSKIIPAIEGLVFPYFTNCHEALKEDGR
FGDYIRALRQHLQYVLREGICLFPDGGWKISSTSNNSWLSKIYLCQFIARHILGWE
WDEQAKRADAAHVAWLTHPTLSIWSWSDQIIAGENYRSKYYPRGVTSILWLEE
GE*
MSKITFPKDFIWGSATAAYQIEGAYNEDGKGESIWDRFSHTPGNIADGHTGDVA
CDHYHRYEEDIKIMKEIGIKSYRFSISWPRIFPEGTGKLNQKGLDFYKRLTNLLLE
NGIMPAITLYHWDLPQKLQDKGGWKNRDTTDYFTEYSEVIFKNLGDIVPIWFTH
NEPGVVSLLGHFLGIHAPGIKDLRTSLEVSHNLLLSHGKAVKLFREMNIDAQIGIA
LNLSYHYPASEKAEDIEAAELSFSLAGRWYLDPVLKGRYPENALKLYKKKGIEL
SFPEDDLKLISQPIDFIAFNNYSSEFIKYDPSSESGFSPANSILEKFEKTDMGWIIYPE
GLYDLLMLLDRDYGKPNIVISENGAAFKDEIGSNGKIEDTKRIQYLKDYLTQAHR
AIQDGVNLKAYYLWSLLDNFEWAYGYNKRFGIVHVNFDTLERKIKDSGYWYKE
VIKNNGF*

75

52

1

Q09L
Z0

Geobacillus
stearothermo
philus
(XYNB2,
79.8 kDa)

P2620
8

Clostridium
thermocellu
m
(BGLA, 51.5
kDa)

5.2.4

Enzyme expression and purification

Detailed information is described in Chapter 3 and in a paper published by Gao et al[198].
5.2.5

Strategy for enzyme screening on realistic lignocellulosic substrates

A simple four-step strategy was applied for screening and comparing activities of novel glycosyl
hydrolases to develop enzyme mixtures that can efficiently saccharify pretreated lignocellulosic
biomass (Figure 5.1). A typical benchmark enzyme mixture could include fungal cellulases
(CBH I + CBH II + EG I) along with a suitable β-glucosidase (βG). The goal was to compare the
activity of novel enzymes with respect to a defined benchmark on realistic substrates like
pretreated cellulosic biomass. The first step was to characterize the type of enzyme in order to
assign it to a specific GH family (e.g. pNP-glycoside based activity assays and glycosyl
hydrolase family determination based on amino acid sequence similarity). The second step was
doping the new enzyme/s along with the benchmark mixture to determine the effect on
digestibility of pretreated biomass. It may be necessary to swap the corresponding type of
enzyme from the benchmark mixture before adding new enzymes to compare relative
improvements. This iterative method allows one to determine the most efficient enzyme/enzyme
mixtures that have high activity on pretreated lignocellulosic biomass and avoid the pitfall of
screening individual enzymes on unrealistic substrates (e.g. CMC, pNP-glycosides) [114]. Once
a minimal enzyme mixture has been defined it should be possible to further reduce enzyme
dosage by optimizing the relative ratios of the enzymes in the mixture to maximize glucan and
xylan digestibility [71].

76

1. Characterize enzyme (ENEW)
Protein sequence similarity, Activity assays

2. Dope new enzyme to defined benchmark mixture
Benchmark: CBH I + CBH II + EG I + bG
Dope mixture: Benchmark + ENEW

3. Swap new enzyme with respective family type
Swap EG I from Benchmark with ENEW
Swap mixture: CBH I + CBH II + ENEW + βG

4. Optimize relative ratios and total protein loading for
new enzymes in mixture to maximize digestibility

Figure 5.1 Four-step strategy for screening glycosyl hydrolases and developing novel
enzyme mixtures to maximize digestibility of pretreated lignocellulose.
5.2.6

Enzyme activity assays

Detailed information is described in 4.2.6.
5.2.7

Enzymatic hydrolysis of pretreated biomass

Detailed information is described in 4.2.6.
5.2.8

Glucose and xylose assays

Detailed information is described in 4.2.8.

77

5.3 Results and discussions
5.3.1

Specific activities of bacterial and fungal enzymes

The enzymes were tested for their activity on different substrates at pH 5 (Table 5.2). LC1 and
LC2 have significant pNP-cellobioside and CMC activity. Although both of the endocellulases
were found to have significant activity on pNPC, their CMC activity was slightly lower or
comparable to EG I. LX1 and LX2 were found to have much higher xylanase activity than EG I,
though earlier work has reported another Geobacillus xylanase (with 88% similarity to LX1) to
have lost 40-60% activity at pH 5 [66]. Recent work with a fungal endo-xylanase isolated from
Trichoderma has shown that the Geobacillus enzyme has 3-5 fold higher activity on oat spelt
xylan under identical conditions [71]. LβX was found to have high xylosidase activity but poor
α-arabinofuranosidase activity, comparable to what has been reported earlier [199]. The bacterial
β-glucosidase (LβG) had significantly lower cellobiose activity than its fungal counterpart (βG).
No noticeable activity on Avicel was detected for any of the bacterial enzymes compared to the
fungal cellulases.
Table 5.2 Activity assay data for bacterial and fungal enzymes.
Activity Units
CBH
II
0.03
-

EG I

βG

130.1
143.8
320.3
15.1
-

CBH
I
1.4
7.4
0.02
-

44.6
19.3
0.01
6.7
5.1

2470
4150
9.52
-

31.1

-

-

-

125

LC1
pNPC *
pNPL *
pNPG *
pNPAf *
pNPX *
Avicel **
CMC **
Xylan **
Cellobiose
**

LC2

LX1

LX2

LβX

LβG

79.6
207.7
6.6
-

378.8
353.1
1.1
-

11.1
0.9
19.3

65.6
8.3
4.2
24.4

1.5
9.4
1545
-

-

-

-

-

7.8

78

One unit of activity on pNP based substrates (*) is equivalent to one nmol of pNP (pnitrophenol) released/mg enzyme/min.
One unit of activity on Avicel/Xylan/CMC/Cellobiose (**) is equivalent to one µmol of
glucose equivalent released/mg enzyme/min.
Where; “-“ stands for no detectable activity; pNPC (pNP-β-D-cellobioside); pNPL (pNP-βD-lactopyranoside);
pNPG
(pNP-β-D-glucopyranoside);
pNPAf
(pNP-α-Larabinofuranoside); pNPX (pNP-β-D-xylopyranoside); CMC (Carboxymethyl cellulose).
Figure 5.2 depicts the hydrolysis yields on AFEX corn stover for all 6 bacterial enzymes added
together as a mixture at varying pH and temperatures. The enzyme loading was 4 mg/g glucan
each for LC1, LC2, LX1 and LX2; 2 mg/g glucan each for LβX and LβG. At pH 6.5 and 50 °C,
the xylose yield was approximately 50%, suggesting the high hemicellulase activity for the
enzymes. However, the glucan conversion was significantly lower (< 5%). The activity assays
showed that none of the bacterial enzymes had any significant activity on Avicel (Table 5.2).
Although the bacterial enzymes cloned belonged to thermophilic microbes, hydrolysis yields at
o

o

70 C were lower compared to 50 C. It is possible that the enzymes lost activity at high
temperature during the prolonged incubation (24 hours). Since the bacterial and fungal enzymes
have a different working pH ranges, a mixture of both enzymes was tested on pH 6.5 and pH 4.5.
For fungal enzymes, the optimal pH was found to be at 4.5-5 (data not shown). When tested at
pH 6.5, significant loss in activity was observed for the fungal enzymes. For an equimass
mixture of CBH I, CBH II and EG I, the glucan hydrolysis yield decreased to 10% (pH 6.5)
compared to 60% at pH 4.5 (24 hrs hydrolysis, data not shown).
Interestingly, the bacterial β-xylosidase has been reported earlier to retain about 40% activity at
pH 5 vs. pH 6.5 [199]. We noticed only a 25% loss in activity based on the overall xylan
conversions for AFEX corn stover (Figure 5.3). This would suggest that optimizing the bacterial

79

hemicellulases at pH 4.5-5 along with fungal cellulases would be possible considering the
significant activity retained at acidic pH.

100%
glucose
Conversion

75%

xylose

50%

25%

0%
pH 6.5, 50°C pH 4.5, 50°C pH 6.5, 65°C pH 4.5, 70°C
Figure 5.2 Percent glucan (blue bar) and xylan (red bar) conversion after 24 hours
hydrolysis of AFEX treated corn stover. Six bacterial enzymes were added together as a
mixture at varying pH and temperature. Each enzyme mixture contains both bacterial
cellulases and hemicellulases (4 mg/g glucan each for LC1, LC2, LX1 and LX2; 2 mg/g
glucan each for LβX and LβG).

5.3.2

Doping and swapping bacterial/fungal cellulases

The experimental design for the doping and swapping experiments is shown in Table 5.3, which
o

was conducted under specific assay conditions (pH 4.5-5.0, 50 C, 24 hrs) using AFEX treated
corn stover. From Figure 5.3A, experiments #A-B show that swapping of LβG with βG does not
significantly increase the glucose or xylose yield. From the results of experiment #G it can be

80

observed that LβG showed lower glucan conversion even at much higher enzyme loadings
(Figure 5.3B and Table 5.3). Experiments #F-J also indicate that the bacterial β-glucosidase has
much lower activity compared to its fungal counterpart. The current batch of bacterial enzymes
does not possess substantial exo-cellulase activity to hydrolyze AFEX treated corn stover.
Therefore, purified fungal cellulases (CBH I, CBH II, EG I) were doped into the enzyme mix in
order to further enhance the glucose yield (Experiments #C-E). Doping any of the three fungal
cellulases increased the glucan conversion to around 20%. Interestingly, #E has a higher xylose
yield possibly due to cross-activity of EG I on xylan. Doping LC1, LC2 or both together into a
fungal mixture did not significantly improve either the glucose or xylose hydrolysis yield
(Experiments #K-M). The above results demonstrate that bacterial cellulases (LC1, LC2, LβG)
do not significantly improve the digestibility of pretreated biomass compared to fungal cellulases
(CBH I, CBH II, βG), despite the fact that the bacterial enzymes were found to have significant
activity on artificial substrates (like CMC and pNP-glycosides). Wilson et al. have shown
synergism between certain bacterial endo-glucanases and fungal exo-glucannases on filter paper,
but the overall digestions were still quite low [130]. It is possible that due to lack of suitable exocellulase activity the bacterial cellulases (LC1 and LC2) are currently unable to completely
hydrolyze the substrate. Previous results for Clostridium thermocellum β-glucosidase have also
revealed that gene functions encoding for hydrolytic activity on MU-glucoside and/or cellobiose
are associated closely on the chromosome [130].

81

Table 5.3 Mixtures of bacterial and fungal enzymes tested on AFEX treated corn stover. The experimental results for these
enzyme combinations are shown in Figure 5.3.

A

B

C

#
A
B
C
D
E
F
G
H
I
J
K
L
M
O

Mix type
Control
Swap
Dope
Dope
Dope
Swap
Swap
Benchmark
Swap/Dope
Dope
Dope
Dope
Dope
Dope

P
Q

Dope
Dope

R

Dope

LC1
4
4
4
4
4

4
4

Individual enzyme loading (mg/g glucan)
LC2
LX1
LX2
LβG
LβX
CBH I
4
4
4
2
2
4
4
4
2
4
4
4
2
4
2
4
4
4
4
4
4
2
2
4
10
4
4
2
2
4
2
4
4
4
4
4
4
4
4
4
4
4
4
4
4

4

82

2

4

CBH II

EG I

4

βG
2
2
2
2

4
4
4
4
4
4
4
4
4
4
4

4
4
4
4
4
4
4
4
4
4
4
4

2
2
2
2
2
2
2

4

4

2

2

5.3.3

Doping of bacterial hemicellulases to fungal benchmark mixture

Although bacterial cellulases (LC1, LC2 and LβG) did not work effectively on their own or work
synergistically with fungal enzymes, the bacterial hemicellulases (LX1, LX2 and LβX) were
found to have significant activity on pretreated corn stover (Experiments #O-R). As shown in
Figure 5.3C (experiment design shown in Table 5.3), doping LX1 and LX2 to the fungal
benchmark mixture helps increase the xylose and glucose yield. Hydrolyzing xylan enhances the
accessibility of the cellulases to the residual cellulose microfibrils and results in higher glucan
conversions. When LX1 and LX2 were doped together (Experiment #Q), glucan conversion
increased to 70% while no noticeable increase was seen for xylose (Experiments #O-P). Addition
of LβX (Experiment #R), helped increase the xylan conversion substantially (71% glucan and 76%
xylan conversion). In order to enhance both glucan and xylan conversion, LβX is important to
hydrolyze soluble xylan based oligosaccharides which are inhibitors of endoxylanases and
cellulases. There have been reports on increased conversions on both xylan and glucan by
supplementation of hemicellulases [139]. In order to completely digest the xylan fraction, βxylosidase is indispensible to hydrolyze xylo-oligosaccharides, especially xylobiose to
monomeric xylose [200]. Previous results have also shown that addition of bacterial
hemicellulases to fungal cellulases, results in increasing both glucose and xylose yields [201].

83

A

100%

Glucose

75%
Conversion

Xylose
50%

25%

0%
A

B

C

D

E

Figure 5.3 Percent glucan (blude bar) and xylan (red bar) conversions after 24 hours
hydrolysis of AFEX treated corn stover. Enzyme mixtures used for experimental data sets
in panel A (A to E), panel B (F to M) and panel C (H, O to R) are based on protein
compositions listed in Table 5.3.

84

Figure 5.3 (cont’d)

B 100%
Glucose
Xylose

Conversion

75%

50%

25%

0%
F

G

H

I

J

K

L

M

C
100%
Glucose
Xylose

Conversion

75%

50%

25%

0%
H

O

P
85

Q

R

5.3.4

Enzyme mixture optimization

Previous results have demonstrated the synergistic interactions between fungal cellulases (CBH I,
CBH II, EG I and βG) and bacterial hemicellulases (LX1, LX2 and LβG). However, individual
enzyme ratios need to be optimized to allow further increase in glucan and xylan conversions. In
order to do this, 73 different enzyme combinations were tested in duplicates (standard deviations
were less than 5% for the replicates) and the average hydrolysis yields for both glucan and xylan
were determined for three different protein loadings (Table 5.4). βG was loaded at a 10% (mass
ratio) of the total remaining enzymes to ensure complete hydrolysis of cellobiose [125]. CBH I
and CBH II were added at more than 20% (total protein excluding βG) in all mixtures to ensure
sufficient cellulase activity. EG I added was at least 10% of the mixture. Bacterial LX1 and LX2
together were always more than 5% of total enzyme loading while bacterial LβX was greater
than 1%. All of the above constraints were based on the fact that cellobiohydrolase,
endoglucanase, β-glucosidase, endoxylanase and β-xylosidase are indispensible for an efficient
enzyme cocktail [71]. Deconstruction of crystalline cellulose is the major limiting step towards
complete hydrolysis, and hence requires a significant amount of cellulase. It is clear that
increasing the bacterial hemicellulases loading beyond 10 mg/g of glucan does not significant
increase the xylan conversion. Using the current cocktail of enzymes the xylan conversion could
not exceed 75% conversion even when glucan conversion was over 90%. It is possible that other
hemicellulases (e.g. α-arabinofuranosidase, α-glucuronidase) are necessary to further increase
the xylan conversion. Without suitable complementary hemicellulases, xylan conversion is a
bottleneck and increasing the total enzyme loading alone would not result in 100% xylan
conversion.

86

Table 5.4 24 hours glucan and xylan hydrolysis yields of AFEX treated corn stover by
various enzyme mixtures at three different total protein loadings. βG was loaded at 10
(mass loading) of all other proteins.
Enzymes ratio (%)
#

CBH I

CBH II

EG I

LX1

LX2

LβX

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34

20.0
84.0
0.0
0.0
84.0
10.0
0.0
0.0
52.0
20.0
0.0
0.0
0.0
42.0
51.0
52.0
10.0
10.0
0.0
9.0
52.0
0.0
20.0
52.0
9.0
52.0
84.0
0.0
42.0
51.0
20.0
9.0
20.0
0.0

0.0
0.0
52.0
20.0
0.0
10.0
84.0
20.0
0.0
0.0
52.0
20.0
84.0
42.0
9.0
0.0
10.0
10.0
20.0
51.0
0.0
20.0
0.0
0.0
19.0
0.0
0.0
52.0
42.0
9.0
0.0
19.0
0.0
52.0

10.0
10.0
42.0
10.0
10.0
74.0
10.0
10.0
42.0
10.0
10.0
10.0
10.0
10.0
18.0
42.0
10.0
10.0
74.0
18.0
10.0
10.0
10.0
10.0
18.0
10.0
10.0
10.0
10.0
18.0
10.0
18.0
42.0
10.0

0.0
5.0
5.0
34.5
2.5
5.0
0.0
0.0
0.0
0.0
37.0
0.0
2.5
5.0
5.3
5.0
69.0
0.0
5.0
7.8
37.0
69.0
34.5
5.0
5.3
0.0
0.0
0.0
0.0
7.8
69.0
7.8
0.0
5.0

37.0
0.0
0.0
34.5
2.5
0.0
5.0
37.0
5.0
69.0
0.0
69.0
2.5
0.0
7.8
0.0
0.0
69.0
0.0
5.3
0.0
0.0
34.5
0.0
7.8
5.0
5.0
5.0
5.0
5.3
0.0
5.3
37.0
0.0

33.0
1.0
1.0
1.0
1.0
1.0
1.0
33.0
1.0
1.0
1.0
1.0
1.0
1.0
9.0
1.0
1.0
1.0
1.0
9.0
1.0
1.0
1.0
33.0
41.0
33.0
1.0
33.0
1.0
9.0
1.0
41.0
1.0
33.0

87

30 mg/g glucan
Glucan Xylan
Yield
yield
(%)
(%)
47.6
49.3
70.1
47.3
56.5
49.2
38.1
46.3
60.6
38.3
89.0
54.1
45.4
36.9
31.3
44.5
68.0
44.6
51.4
50.2
52.0
48.1
40.4
48.7
48.2
41.0
81.2
46.8
83.9
56.1
71.3
47.6
80.1
54.4
67.2
48.5
44.8
40.8
86.2
60.2
63.7
47.9
41.1
49.6
53.1
50.6
65.7
55.3
79.6
56.5
53.3
45.7
64.6
41.0
37.1
39.9
78.3
43.6
90.2
60.2
52.7
52.0
81.2
60.0
65.1
47.0
45.3
48.8

Table 5.4 (cont’d)
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73

0.0
20.0
0.0
0.0
9.0
19.0
19.0
9.0
20.0
20.0
10.0
0.0
0.0
0.0
0.0
20.0
9.0
20.0
9.0
19.0
20.0
0.0
20.0
20.0
19.0
10.0
0.0
52.0
20.0
20.0
19.0
0.0
0.0
0.0
9.0
18.0
19.0
0.0
10.0

20.0
0.0
20.0
20.0
51.0
9.0
9.0
19.0
0.0
0.0
10.0
52.0
20.0
20.0
20.0
0.0
19.0
0.0
19.0
9.0
0.0
20.0
0.0
0.0
9.0
10.0
20.0
0.0
0.0
0.0
9.0
84.0
52.0
20.0
19.0
18.0
9.0
20.0
10.0

74.0
74.0
10.0
42.0
18.0
50.0
18.0
50.0
10.0
10.0
10.0
42.0
10.0
42.0
10.0
10.0
50.0
74.0
18.0
18.0
42.0
42.0
74.0
10.0
50.0
10.0
10.0
10.0
42.0
42.0
18.0
10.0
10.0
74.0
18.0
26.0
18.0
42.0
74.0

2.5
2.5
37.0
0.0
5.3
5.3
5.3
7.8
0.0
37.0
0.0
0.0
0.0
37.0
2.5
5.0
5.3
5.0
39.8
7.8
0.0
0.0
0.0
2.5
7.8
5.0
5.0
0.0
5.0
37.0
5.3
5.0
0.0
0.0
5.3
10.5
39.8
5.0
0.0

88

2.5
2.5
0.0
37.0
7.8
7.8
7.8
5.3
5.0
0.0
5.0
5.0
5.0
0.0
2.5
0.0
7.8
0.0
5.3
5.3
5.0
5.0
5.0
2.5
5.3
0.0
0.0
37.0
0.0
0.0
39.8
0.0
37.0
5.0
39.8
10.5
5.3
0.0
5.0

1.0
1.0
33.0
1.0
9.0
9.0
41.0
9.0
65.0
33.0
65.0
1.0
65.0
1.0
65.0
65.0
9.0
1.0
9.0
41.0
33.0
33.0
1.0
65.0
9.0
65.0
65.0
1.0
33.0
1.0
9.0
1.0
1.0
1.0
9.0
17.0
9.0
33.0
1.0

44.5
67.1
37.5
45.7
81.2
81.9
79.5
90.0
42.6
53.5
64.0
49.5
33.1
48.4
33.0
46.7
77.7
64.8
83.6
85.3
57.2
43.5
67.0
48.8
94.7
74.7
33.0
63.5
57.3
58.4
76.5
49.9
44.5
46.8
74.9
77.3
79.0
45.4
82.4

40.4
47.5
48.2
49.4
56.0
54.1
56.8
59.3
47.2
55.1
49.7
42.3
42.8
50.3
42.7
50.3
53.6
45.8
59.7
60.0
49.5
50.4
48.7
52.7
62.9
57.9
44.7
49.6
49.5
46.3
53.8
44.2
45.9
43.3
53.4
53.6
54.8
53.3
50.1

Table 5.4 (cont’d)
Enzymes ratio (%)
#

CBH I

CBH II

EG I

LX1

LX2

LβX

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34

20.0
84.0
0.0
0.0
84.0
10.0
0.0
0.0
52.0
20.0
0.0
0.0
0.0
42.0
51.0
52.0
10.0
10.0
0.0
9.0
52.0
0.0
20.0
52.0
9.0
52.0
84.0
0.0
42.0
51.0
20.0
9.0
20.0
0.0

0.0
0.0
52.0
20.0
0.0
10.0
84.0
20.0
0.0
0.0
52.0
20.0
84.0
42.0
9.0
0.0
10.0
10.0
20.0
51.0
0.0
20.0
0.0
0.0
19.0
0.0
0.0
52.0
42.0
9.0
0.0
19.0
0.0
52.0

10.0
10.0
42.0
10.0
10.0
74.0
10.0
10.0
42.0
10.0
10.0
10.0
10.0
10.0
18.0
42.0
10.0
10.0
74.0
18.0
10.0
10.0
10.0
10.0
18.0
10.0
10.0
10.0
10.0
18.0
10.0
18.0
42.0
10.0

0.0
5.0
5.0
34.5
2.5
5.0
0.0
0.0
0.0
0.0
37.0
0.0
2.5
5.0
5.3
5.0
69.0
0.0
5.0
7.8
37.0
69.0
34.5
5.0
5.3
0.0
0.0
0.0
0.0
7.8
69.0
7.8
0.0
5.0

37.0
0.0
0.0
34.5
2.5
0.0
5.0
37.0
5.0
69.0
0.0
69.0
2.5
0.0
7.8
0.0
0.0
69.0
0.0
5.3
0.0
0.0
34.5
0.0
7.8
5.0
5.0
5.0
5.0
5.3
0.0
5.3
37.0
0.0

33.0
1.0
1.0
1.0
1.0
1.0
1.0
33.0
1.0
1.0
1.0
1.0
1.0
1.0
9.0
1.0
1.0
1.0
1.0
9.0
1.0
1.0
1.0
33.0
41.0
33.0
1.0
33.0
1.0
9.0
1.0
41.0
1.0
33.0

89

15mg/g glucan
Glucan
Xylan
Yield
yield
(%)
(%)
31.8
41.6
44.7
33.0
37.7
35.9
28.4
39.3
44.0
30.6
60.7
37.5
30.3
26.0
21.8
38.8
49.4
31.5
31.6
36.3
34.8
38.7
26.1
34.8
33.0
29.1
66.5
35.2
69.5
50.3
55.4
37.6
54.9
40.8
51.2
37.8
34.2
35.9
59.5
46.8
42.6
39.3
27.8
38.3
33.7
40.7
38.5
43.2
62.5
46.8
34.2
36.8
39.7
25.2
26.8
35.0
61.8
28.1
71.6
51.0
32.2
40.3
58.4
46.8
44.5
38.6
29.5
40.0

Table 5.4 (cont’d)
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73

0.0
20.0
0.0
0.0
9.0
19.0
19.0
9.0
20.0
20.0
10.0
0.0
0.0
0.0
0.0
20.0
9.0
20.0
9.0
19.0
20.0
0.0
20.0
20.0
19.0
10.0
0.0
52.0
20.0
20.0
19.0
0.0
0.0
0.0
9.0
18.0
19.0
0.0
10.0

20.0
0.0
20.0
20.0
51.0
9.0
9.0
19.0
0.0
0.0
10.0
52.0
20.0
20.0
20.0
0.0
19.0
0.0
19.0
9.0
0.0
20.0
0.0
0.0
9.0
10.0
20.0
0.0
0.0
0.0
9.0
84.0
52.0
20.0
19.0
18.0
9.0
20.0
10.0

74.0
74.0
10.0
42.0
18.0
50.0
18.0
50.0
10.0
10.0
10.0
42.0
10.0
42.0
10.0
10.0
50.0
74.0
18.0
18.0
42.0
42.0
74.0
10.0
50.0
10.0
10.0
10.0
42.0
42.0
18.0
10.0
10.0
74.0
18.0
26.0
18.0
42.0
74.0

2.5
2.5
37.0
0.0
5.3
5.3
5.3
7.8
0.0
37.0
0.0
0.0
0.0
37.0
2.5
5.0
5.3
5.0
39.8
7.8
0.0
0.0
0.0
2.5
7.8
5.0
5.0
0.0
5.0
37.0
5.3
5.0
0.0
0.0
5.3
10.5
39.8
5.0
0.0

90

2.5
2.5
0.0
37.0
7.8
7.8
7.8
5.3
5.0
0.0
5.0
5.0
5.0
0.0
2.5
0.0
7.8
0.0
5.3
5.3
5.0
5.0
5.0
2.5
5.3
0.0
0.0
37.0
0.0
0.0
39.8
0.0
37.0
5.0
39.8
10.5
5.3
0.0
5.0

1.0
1.0
33.0
1.0
9.0
9.0
41.0
9.0
65.0
33.0
65.0
1.0
65.0
1.0
65.0
65.0
9.0
1.0
9.0
41.0
33.0
33.0
1.0
65.0
9.0
65.0
65.0
1.0
33.0
1.0
9.0
1.0
1.0
1.0
9.0
17.0
9.0
33.0
1.0

30.4
45.9
28.0
30.5
64.7
70.2
61.2
64.2
24.4
37.9
43.6
35.7
21.3
33.6
21.9
27.2
61.0
44.2
61.8
62.4
38.6
29.7
42.6
30.0
71.8
46.0
22.4
40.0
40.3
44.8
60.8
33.1
29.7
31.8
59.9
65.5
58.5
30.2
61.8

33.0
33.4
46.4
36.8
49.7
42.5
48.8
47.1
36.1
46.3
39.3
29.5
35.6
41.2
37.6
40.7
46.9
34.8
49.2
48.7
42.7
42.1
32.7
40.0
54.5
45.4
39.0
35.2
47.4
41.7
47.8
31.1
33.1
30.3
49.0
49.4
48.2
45.4
36.1

Table 5.4 (cont’d)
Enzymes ratio (%)
#

CBH I

CBH II

EG I

LX1

LX2

LβX

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34

20.0
84.0
0.0
0.0
84.0
10.0
0.0
0.0
52.0
20.0
0.0
0.0
0.0
42.0
51.0
52.0
10.0
10.0
0.0
9.0
52.0
0.0
20.0
52.0
9.0
52.0
84.0
0.0
42.0
51.0
20.0
9.0
20.0
0.0

0.0
0.0
52.0
20.0
0.0
10.0
84.0
20.0
0.0
0.0
52.0
20.0
84.0
42.0
9.0
0.0
10.0
10.0
20.0
51.0
0.0
20.0
0.0
0.0
19.0
0.0
0.0
52.0
42.0
9.0
0.0
19.0
0.0
52.0

10.0
10.0
42.0
10.0
10.0
74.0
10.0
10.0
42.0
10.0
10.0
10.0
10.0
10.0
18.0
42.0
10.0
10.0
74.0
18.0
10.0
10.0
10.0
10.0
18.0
10.0
10.0
10.0
10.0
18.0
10.0
18.0
42.0
10.0

0.0
5.0
5.0
34.5
2.5
5.0
0.0
0.0
0.0
0.0
37.0
0.0
2.5
5.0
5.3
5.0
69.0
0.0
5.0
7.8
37.0
69.0
34.5
5.0
5.3
0.0
0.0
0.0
0.0
7.8
69.0
7.8
0.0
5.0

37.0
0.0
0.0
34.5
2.5
0.0
5.0
37.0
5.0
69.0
0.0
69.0
2.5
0.0
7.8
0.0
0.0
69.0
0.0
5.3
0.0
0.0
34.5
0.0
7.8
5.0
5.0
5.0
5.0
5.3
0.0
5.3
37.0
0.0

33.0
1.0
1.0
1.0
1.0
1.0
1.0
33.0
1.0
1.0
1.0
1.0
1.0
1.0
9.0
1.0
1.0
1.0
1.0
9.0
1.0
1.0
1.0
33.0
41.0
33.0
1.0
33.0
1.0
9.0
1.0
41.0
1.0
33.0

91

10 mg/g glucan
Glucan
Xylan
Yield
yield
(%)
(%)
26.2
43.9
34.8
26.3
31.9
27.2
23.1
35.5
34.7
21.5
51.4
29.8
25.0
20.1
19.5
40.2
37.5
24.5
20.1
26.1
28.6
31.2
20.9
29.5
25.7
20.6
61.3
28.6
55.9
43.0
45.0
30.0
45.2
34.3
41.9
30.3
23.7
26.1
53.4
44.1
37.4
34.6
21.7
33.8
25.6
33.3
30.0
39.2
53.3
48.3
30.6
36.3
30.0
20.9
20.6
29.9
52.4
20.3
58.8
47.9
25.3
34.0
51.7
50.5
34.3
30.8
24.9
39.7

Table 5.4 (cont’d)
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73

0.0
20.0
0.0
0.0
9.0
19.0
19.0
9.0
20.0
20.0
10.0
0.0
0.0
0.0
0.0
20.0
9.0
20.0
9.0
19.0
20.0
0.0
20.0
20.0
19.0
10.0
0.0
52.0
20.0
20.0
19.0
0.0
0.0
0.0
9.0
18.0
19.0
0.0
10.0

20.0
0.0
20.0
20.0
51.0
9.0
9.0
19.0
0.0
0.0
10.0
52.0
20.0
20.0
20.0
0.0
19.0
0.0
19.0
9.0
0.0
20.0
0.0
0.0
9.0
10.0
20.0
0.0
0.0
0.0
9.0
84.0
52.0
20.0
19.0
18.0
9.0
20.0
10.0

74.0
74.0
10.0
42.0
18.0
50.0
18.0
50.0
10.0
10.0
10.0
42.0
10.0
42.0
10.0
10.0
50.0
74.0
18.0
18.0
42.0
42.0
74.0
10.0
50.0
10.0
10.0
10.0
42.0
42.0
18.0
10.0
10.0
74.0
18.0
26.0
18.0
42.0
74.0

2.5
2.5
37.0
0.0
5.3
5.3
5.3
7.8
0.0
37.0
0.0
0.0
0.0
37.0
2.5
5.0
5.3
5.0
39.8
7.8
0.0
0.0
0.0
2.5
7.8
5.0
5.0
0.0
5.0
37.0
5.3
5.0
0.0
0.0
5.3
10.5
39.8
5.0
0.0

92

2.5
2.5
0.0
37.0
7.8
7.8
7.8
5.3
5.0
0.0
5.0
5.0
5.0
0.0
2.5
0.0
7.8
0.0
5.3
5.3
5.0
5.0
5.0
2.5
5.3
0.0
0.0
37.0
0.0
0.0
39.8
0.0
37.0
5.0
39.8
10.5
5.3
0.0
5.0

1.0
1.0
33.0
1.0
9.0
9.0
41.0
9.0
65.0
33.0
65.0
1.0
65.0
1.0
65.0
65.0
9.0
1.0
9.0
41.0
33.0
33.0
1.0
65.0
9.0
65.0
65.0
1.0
33.0
1.0
9.0
1.0
1.0
1.0
9.0
17.0
9.0
33.0
1.0

24.8
35.4
23.1
24.0
61.0
63.3
46.9
56.9
18.2
30.2
32.9
32.8
16.9
28.7
16.5
20.4
60.1
37.1
51.6
54.8
29.7
22.6
31.6
24.5
63.1
35.4
18.2
34.1
29.6
37.1
52.8
28.4
24.5
26.0
47.4
58.6
52.2
24.9
43.7

25.7
25.6
46.5
27.5
43.4
52.2
39.2
37.3
34.2
45.3
35.9
26.4
33.8
35.6
33.2
35.7
48.8
31.2
48.3
44.9
42.2
38.7
25.7
39.6
52.3
43.5
38.6
28.9
40.1
36.6
47.9
25.6
25.8
24.1
42.6
49.9
49.3
40.5
25.1

Glucan and xylan hydrolysis results for all mixtures are shown in Figure 5.4 as a scatter plot. The
relative ratio of the individual enzymes significantly affected the overall sugar yield. At 10 mg/g
glucan loading, the highest glucan conversion is 63.1% while the lowest is 20.6%. The highest
xylan conversion is 52.3% while the lowest is 20.1%, at the same protein loading. Similarly,
major differences in overall conversions can be seen for other protein loadings as well. One of
the best mixtures resulting in the highest glucan and xylan conversions contained 19% CBH I, 9%
CBH II, 50% EG I, 7.8% LX1, 5.3% LX2 and 9.0% LβX.

75%
10 mg enzyme/g glucan
15 mg enzyme/g glucan

Xylan conversion

30 mg enzyme/g glucan
50%

25%

0%
0%

25%
50%
Glucan conversion

75%

100%

Figure 5.4 Glucan (X-axis) versus xylan (Y-axis) conversion after 24 hours hydrolysis of
AFEX treated corn stover. Hydrolysis results from three different total enzyme loadings
(inclusive of CBH I + CBH II + EG I + LX 1 + LX 2 + LβX, as listed in
β

93

Table 5.4) are depicted by green triangles (30 mg/g glucan), red squares (15 mg/g glucan)
and blue diamonds (10 mg/g glucan). An additional loading of 3, 1.5 and 1 mg/g glucan of
βG was supplemented in each case, respectively.
5.3.5

Relationship between glucan and xylan conversions

Glucan and xylan conversion for various combinations of enzymes at three different total
enzyme loadings are shown in Figure 5.4. The three clusters for different enzyme loadings
demonstrate that at higher enzyme loading, the glucan and xylan conversion is generally higher.
Another interesting phenomenon observed is that at higher enzyme loadings, the shape of the
data point cluster is narrower. At lower enzyme loadings, the data points are more scattered. By
applying linear regression on xylan conversions vs. corresponding glucan conversion for various
enzyme mixtures, a linear relationship between the two variables is confirmed (Table 5.5). The P
values are close to 0, indicating that the linear relationship has statistical significance. When the
2

total enzyme loading was increased (from 10 to 30 mg/g glucan) the R (Coefficient of
determination) value increases as well. This validates our visual interpretation of the shape of the
data cluster at low vs. high enzyme loadings.
Table 5.5 Linear regression of xylan vs. glucan conversion at three different total enzyme
loadings.
Xylan conversion = Constant + A*glucan conversion
Constant
A
Enzyme loading (mg/g glucan)
Coefficient
P
Coefficient
P
10
0.24347
0.000
0.3061
<0.001
15
0.29395
0.000
0.23899
<0.001
0.24774
<0.001
30
0.34521
0.000

R

2

0.218
0.262
0.451

CBH I and CBH II are both indispensible for efficient hydrolysis. For all 3 varying enzyme
loadings, if the mixture does not contain CBH I, the glucan conversions are quite low. When

94

both CBH I and CBH II are included, higher glucan conversions are possible (>90% glucan
conversion at 30 mg/g glucan enzyme loading). Xylan conversion is not markedly affected by
the presence of either CBH I or II. Xylan conversion tends to be slightly higher (5-10%) at
higher enzyme loadings when both CBH’s are present. LX1 has slightly higher specific activity
compared to LX2 on AFEX treated corn stover.
Figure 5.5 is helpful in visually summarizing the optimal regions of enzyme ratios for
maximizing both glucan and xylan digestibility. Different clusters of data (based Table 5.4) were
separated based on the overall ratio of cellulases (I), xylanases (II) and βX (III). The three
enzyme loadings were plotted as insets (a), (b) and (c) representing 10, 15 and 30 mg/g glucan
enzyme loading, respectively. At high enzyme loadings, the higher glucan and xylan yielding
data points aggregate closely compared to the lower enzyme loading. This suggests that glucan
and xylan yields are more sensitive to individual enzyme ratios at lower enzyme loading. At high
cellulase loading (94%), the hemicellulase loading is much lower and both glucan and xylan
yields are relatively lower. This further confirms our previous assumption that in order to
maximize glucan yield, higher xylan hydrolysis yields are desirable. On the other hand, for
higher hemicellulase loading (>37%), xylan conversion is slightly lower while glucan
conversions drop significantly. Similar trends for βX at around 9% loading of total protein mass
ratio are seen as well. To achieve high conversions of glucan and xylan, 78% cellulases (CBH I,
CBH II and EG I), 13% xylanase (LX1 and LX2) and 9% βX seems to be optimal. The optimal
cellulase loading (total of 78%; total protein mass basis) for both CBH I and CBH II ranges from
9-51%; while EG I ranges from 10-50%.

95

70%

I.a
Xylan conversion

60%
50%
40%
30%
20%
10%
0%
0%

20%

40%

60%

80%

100%

80%

100%

Glucan conversion
70%

I.b

Xylan conversion

60%
50%
40%
30%
20%
10%
0%
0%

20%

40%

60%

Glucan conversion
Figure 5.5 Glucan (X-axis) versus xylan (Y-axis) conversion after 24 hours hydrolysis of
AFEX treated corn stover for varying relative ratios of cellulases (panel I), xylanases (panel
II) and β-xylosidase (panel III) at three different total enzyme loadings (a, b and c
correspond to 10, 15 and 30 mg/g glucan enzyme loading, respectively).

96

Figure 5.5 (cont’d)

70%

I.c
Xylan conversion

60%
50%
40%
30% cellulase
46% cellulase
62% cellulase
78% cellulase
94% cellulase

30%
20%
10%
0%
0%

20%

40%

60%

80%

100%

Glucan conversion
70%

II.a
Xylan conversion

60%
50%
40%
30%
20%
10%
0%
0%

20%

40%

60%

Glucan conversion

97

80%

100%

Figure 5.5 (cont’d)

70%

II.b

Xylan conversion

60%
50%
40%
30%
20%
10%
0%
0%

20%

40%

60%

80%

100%

80%

100%

Glucan conversion
70%

II.c

Xylan conversion

60%
50%
40%
30%
20%

5% LX1+LX2
13% LX1+LX2
>37% LX1+LX2

10%
0%
0%

20%

40%
60%
Glucan conversion

98

Figure 5.5 (cont’d)

70%

III.a

Xylan conversion

60%
50%
40%
30%
20%
10%
0%

0%

20%

40%

60%

80%

100%

80%

100%

Glucan conversion
70%

III.b
Xylan conversion

60%
50%
40%
30%
20%
10%
0%
0%

20%

40%

60%

Glucan conversion

99

Figure 5.5 (cont’d)

70%

III.c
Xylan conversion

60%
50%
40%
30%
1% bX

20%

9% bX
10%

>33% bX

0%
0%

20%

40%

60%

80%

100%

Glucan conversion
5.3.6

Discussion

It is interesting to note that the cellobiase activity for the bacterial enzyme (LβG) is significantly
lower than its fungal counterpart (lower activity even at pH 6.5, data not shown). Previous work
has reported anaerobic bacterial enzyme complexes to be easily inhibited by cellobiose [202],
suggesting that cellulosomal activity on cellobiose is relatively poorer (compared to their fungal
counterparts). It is also possible that due to preferred metabolism of cellobiose and the
hydrolyzed oligomers after phosphorylation by the bacterial cell, the activity of β-glucosidase is
relatively poor compared to their fungal counterparts [203].
One of the limitations of hydrolyzing the substrate at pH 5 is the relatively lower activity of these
bacterial enzymes (40-50% of optimum at pH 6.5) reported by Bravman et al. [199]. This would
suggest that in the presence of a suitable bacterial exo/endo-cellulase complex (that has an
100

optimum activity at pH 6.5), it would be possible to further lower the total enzyme loading (and
maximize glucan/xylan conversion). There have also been several reports on the activity of GH
family 43 β-xylosidase, with very few publications on their GH 52 counterparts [199]. This study
is one of the first that reports the activity of GH 52 β-xylosidases on pretreated lignocellulosic
biomass.
5.4 Conclusion
In summary, we have examined the activity of both fungal and bacterial based enzyme mixtures
on a realistic lignocellulosic substrate (i.e. AFEX pretreated corn stover). The results indicate
that certain fungal cellulases and bacterial hemicellulases work synergistically together to
maximize glucan and xylan digestibility. Bacterial xylanases (LX1, LX2 and LβX) can increase
both the glucan and xylan hydrolysis yield when added along with fungal cellulases. Optimized
ratios for individual enzymes are obtained by examining 73 unique enzyme mixture
combinations. Close to 90% glucan and 70% xylan conversion is achieved for the optimal
enzyme combinations. There is a high linear correlation observed between glucose and xylose
hydrolysis yield. Especially at high enzyme loading, this relationship is more obvious. This could
be explained based on the cell wall structural organization and the effect of pretreatment on it.
Within the untreated cell wall ultra-structure, cellulose fibrils are embedded among thin sheaths
of hemicellulose [204]. After pretreatment, the cell wall structure is modified chemically and
ultra-structurally, to substantially enhance enzyme accessibility [14, 33]. Unlike dilute acid
pretreatment, AFEX does not hydrolyze and extract any hemicellulose from the cell wall.
Therefore, hemicellulases are crucial not only to maximize hemicellulose hydrolysis but also
help enhance glucan digestion. The current sets of bacterial hemicellulases are not sufficient to
completely hydrolyze AFEX treated corn stover hemicellulose. In order to further increase xylan

101

conversion greater than 70%, other hemicellulases such as α-arabinofuranosidase and αglucuronidases would be necessary.

102

CHAPTER 6 HEMICELLULASES AND AUXILIARY ENZYMES FOR IMPROVED
CONVERSION OF LIGNOCELLULOSIC BIOMASS TO MONOSACCHARIDES
6.1 Introduction
Previous chapters have allowed us to define the optimum ratio of six core fungal enzymes for
AFEX treated corn stover. The cocktail consisted of cellobiohydrolases I and II (CBH I and CBH
II), endoglucanase I (EG I), β-glucosidase (βG), endoxylanase (EX) and β-xylosidase (βX). More
than 80% of theoretical glucose yield could be achieved using this optimized cocktail. However,
irrespective of the amount of EX and βX loaded, xylose yield never exceeded 56% [71], which
suggests the inclusion of other hemicellulases and/or accessory enzymes is necessary in order to
further increase xylose yields.
Glycosyl hydrolases from bacteria provide a plentiful source of enzymes which have the
potential to be utilized in lignocellulose hydrolysis. Synergism between fungal cellulases and
bacterial xylanases has been recently demonstrated in our previous studies [198]. In this work,
we demonstrate that supplementing fungal cellulases and β-glucosidase (βG) with additional EXs
and other hemicellulases can further increase the yields of glucose and xylose at reduced total
enzyme loadings. Xylanases and two additional debranching hemicellulases from Clostridium
thermocellum, Geobacillus thermodenitrificans, G. stearothermophilus and Dictyoglomus
turgidum (cloned and expressed in Escherichia coli) along with fungal cellulases (CBH I, CBH
II, EG I and βG purified from Trichoderma reesei and Aspergillus niger derived broths) were
evaluated to test their hydrolytic efficacy on AFEX treated corn stover.

103

6.2 Materials and Methods
6.2.1

AFEX pretreatment

Detailed information is described in 4.2.1
6.2.2

Discovery and cloning of LX1, LX2, LβX and LβG

Detailed information is published by Gao et al [198].
6.2.3

Discovery and cloning of L-arabinofuranosidase (LArb)

6.2.4

Discovery and cloning of LX3, LX4, LX5, LX6 and LαGl

Detailed information is published by Gao et al[198, 205]. The protein sequences and strain
sources are listed in Table 6.1.

104

Table 6.1 Amino acid sequences and glycosyl hydrolase families for all six bacterial enzymes.

Notation

LX3

LX4

GH
family

Amino acid sequence

MSGNALRDYAEARGIKIGTCVNYPFYNNSDPTYNSILQREFS
MVVCENEMKFDALQPRQNVFDFSKGDQLLAFAERNGMQM
RGHTLIWHNQNPSWLTNGNWNRDSLLAVMKNHITTVMTHY
KGKIVEWDVANECMDDSGNGLRSSIWRNVIGQDYLDYAFR
YAREADPDALLFYNDYNIEDLGPKSNAVFNMIKSMKERGVPI 10
DGVGFQCHFINGMSPEYLASIDQNIKRYAEIGVIVSFTEIDIRIP
QSENPATAFQVQANNYKELMKICLANPNCNTFVMWGFTDK
YTWIPGTFPGYGNPLIYDSNYNPKPAYNAIKEALMGYHHHH
HH

MKMGKMYEVALVVEGYQSSGKADVTSMTITVGNAPSTSSP
PGPTPEPTPRSAFSKIEAEEYNSLKSSTIQTIGTSDGGSGIGYIE
SGDYLVFNKINFGNGANSFKARVASGADTPTNIQLRLGSPTG
TLIGTLTVASTGGWNNYEEKSCSITNTTGQHDLYLVFSGPVN
IDYFIFDSNGVNPTPTSQPQQGQVLGDLNGDKQVNSTDYTAL
KRHLLNITRLSGTALANADLNGDGKVDSTDLMILHRYLLGII
SSFPRSNPQPSSNPQPSSNPQPTINPNAKLVALTFDDGPDNVL
TARVLDKLDKYNVKATFMVVGQRVNDSTAAIIRRMVNSGH
EIGNHSWSYSGMANMSPDQIRKSIADTNAVIQKYAGTTPKFF
RPPNLETSPTLFNNVDLVFVGGLTANDWIPSTTAEQRAAAVI
NGVRDGTIILLHDVQPEPHPTPEALDIIIPTLKSRGYEFVTLTE
LFTLKGVPIDPSVKRMYNSVP

105

11

Unipr
ot No.

P1047
8

O8711
9

Source, name and
predicted molecular
weight

Truncated Clostridium
thermocellum
(XynZ, 38.0 kDa)
(corresponds to last 324
A.A. in protein of the
mature enzyme)

Truncated
C.
thermocellum
(XynA, 52.1 kDa)
(corresponds to residues
199-683 of the mature
enzyme)

Table 6.1 (cont’d)

LX5

KNKRVLAKITALVVLLGVFFVLPSNISQLYADYEVVHDTFEV
NFDGWCNLGVDTYLTAVENEGNNGTRGMMVINRSSASDGA
YSEKGFYLDGGVEYKYSVFVKHNGTGTETFKLSVSYLDSET
EEENKEVIATKDVVAGEWTEISAKYKAPKTAVNITLSITTDST
VDFIFDDVTITRKGMAEANTVYAANAVLKDMYANYFRVGS
VLNSGTVNNSSIKALILREFNSITCENEMKPDATLVQSGSTNT
NIRVSLNRAASILNFCAQNNIAVRGHTLVWHSQTPQWFFKD
NFQDNGNWVSQSVMDQRLESYIKNMFAEIQRQYPSLNLYAY
DVVNEAVSDDANRTRYYGGAREPGYGNGRSPWVQIYGDNK
10
FIEKAFTYARKYAPANCKLYYNDYNEYWDHKRDCIASICAN
LYNKGLLDGVGMQSHINADMNGFSGIQNYKAALQKYINIGC
DVQITELDISTENGKFSLQQQADKYKAVFQAAVDINRTSSKG
KVTAVCVWGPNDANTWLGSQNAPLLFNANNQPKPAYNAV
ASIIPQSEWGDGNNPAGGGGGGKPEEPDANGYYYHDTFEGS
VGQWTARGPAEVLLSGRTAYKGSESLLVRNRTAAWNGAQR
ALNPRTFVPGNTYCFSVVASFIEGASSTTFCMKLQYVDGSGT
QRYDTIDMKTVGPNQWVHLYNPQYRIPSDATDMYVYVETA
DDTINFYIDEAIGAVAGTVIEGPAPQPTQPPVLLGDVNG

B4BM
E8

LX6

MEIPSLKEVYKDYFPIGAAVSHLNIYTYEDLLKKHFNSLTPEN
QMKWEVIHPKPYVYDFGPADEIVDFAMKNGMKVRGHTLV
WHNQTPGWVYAGTKDEILARLKEHIYEVVGHYKGKVYAW
DVVNEALSDNPNEFLRKAPWYDICGEEVIEKAFIWANEADP
NAKLFYNDYNLEDPIKREKAYQLVKRLKEKGIPIHGVGIQGH 10
WTLAWPTPKMLEDSIKRFSELGVEVQITEFDISIYYDRNENN
NFKVPPDDRIEKQAQLYKQAFEILRKYRGVVTGVTFWGVAD
DYTWLYFWPVRGREDYPLLFDKNHNPKKAFWEIVKFHHHH
HH

B8E34 Dictyoglomus turgidum
6
(Dtur_1647, 38.1 kDa)

106

Truncated
C.
thermocellum
(XynY, 81.4 kDa)
(corresponds to residues
1-736
of
mature
enzyme)

Table 6.1 (cont’d)

LαGl

MAEVKPYNMCWLEYTDLSKYKNKYIKVFENVVVLGGNELN
LPLKELKNFLTFSLNIKPKIFKNTLVKGRNYVLIGRLIEIKKIFK
ESERFEKLLNDEGFIIKRIDIDGNKVLIITAKSYNGIVYGIFNLI
ERLKRGEDIENIDIVSNPSLRFRMLNHWDNLDGSIERGYAGK
SIFFRENKILINERTKDYARLLSSIGVNGVVINNVNVKKKEVE
LITPSYLKKIGELSKIFSAYGIKIY
LSINFASPIYLGGLNTADPLDKRVAVWWKAKVDEIYEYVPD
FGGFLVKADSEFNPGPHMYGRTHADGANMLGEALESYGGF
VIWRAFVYNCLQDWRDTNTDRAKAAYENFKPLDGKFSENVI 67
VQIKYGPMDFQVREPVNPLFGGLEHTNQILELQITQEYTGQQI
HLCYLGTLWKEVLDFDTYAKGEGSKVKEILKGNVFDLKNN
GMAGVSNVGDDINWTGHDLAQANLYTFGALSWNPDERIEE
VVKRWIELTFGDNEKVIKNISYMLLSSHKAYEKYTTPLGLG
WMVNPGHHYGPNPEGYEYSKWGTYHRANYEAIGVDRSSRG
TGYTLQYHSPWREIYDNIETCPEELLLFFHRVPYNYKLKSGK
TLIQTYYDLHFEGVEEAEEIRKKWIELKGEIEDKIYERVLNRL
DIQIEHAKEWRDVINTYFFRRTGIPDEKGRKIYPHHHHHH

107

B8E3
B2

D. turgidum
(Dtur_1714, 79.4 kDa)

6.2.5

Enzyme expression and purification

Detailed information is described in Chapter 3 and in a paper published by Gao et al[198].
6.2.6

Enzymatic hydrolysis of pretreated biomass

Detailed information is described in 4.2.6.
6.2.7

Glucose and xylose assays

Detailed information is described in 4.2.8.
6.3 Results and discussion
6.3.1

Supplementing bacterial hemicellulases along with fungal cellulases

Previous work has shown that Dictyoglomus and Clostridium derived hemicellulases retain
significant activity at pH 4.5-5, whereas Trichoderma derived cellulases lose considerable
activity at pH greater than 5.0 [198]. Hence, all assays were conducted at pH 4.5-5 to maximize
performance of both sets of enzymes. Overall strategies used to obtain an optimized cocktail and
major findings from this study are highlighted in Figure 6.1. From step 2 onwards, each
experimental step was designed and tested based on conclusions from preceding steps. For step 1,
to test performance of the nine different hemicellases on AFEX treated corn stover, all six
xylanases (4 mg/g glucan for each xylanase) and other three accessory enzymes (2 mg/g glucan
for each of LβX, LαGl and LArb, respectively) were grouped and doped together along with the
core cellulases mixture. This avoided the risk of missing any synergism among the
endoxylanases (LXs) and also between LXs and accessory enzymes (LβX, LαGl and LArb).
Core fungal enzymes consisted of CBH I, CBH II and EG I (4 mg/g glucan loading of each
cellulase) plus 2 mg/g glucan βG loading. Previous results have shown the current βG loading is
sufficient to prevent cellobiose build-up at the current glucan loading [71]. The protein mass

108

ratio of CBH I, CBH II and EG I was kept at 1:1:1 in this study since previous results have
demonstrated that this ratio results in optimum activity [198]. The minimum loading of LβX,
LαGl and LArb required was determined after screening for optimal endoxylanase combination.

109

Figure 6.1 Overall conclusions drawn from each step of the process of hemicellulases
optimization in presence of cellulases during hydrolysis of AFEX treated corn stover.

110

From Figure 6.2, core cellulases alone account for 56% glucose yield and minimal (3%) xylose
yield within 24 h hydrolysis. Supplementing xylanases along with core cellulases increased
glucose yield to as high as 83%. However, only 13% xylose yield was achieved. One possible
reason for the improvement in glucan conversion is that xylanase supplementation helps remove
xylan [200] sheathing cellulose fibrils and, hence, increases substrate accessibility [92]. LβX
supplementation allows cleavage of xylo-oligomers to monomeric xylose and hence mixtures
containing LβX have higher monomeric xylose yield. The α-arabinofuranosidase removes
arabinose side-chains, while α-glucuronidase cleaves the α-1,2-glycosidic bond of the 4-Omethyl-D-glucuronic acid side chain from the xylan backbone [29, 69]. Adding LArb and LαGl
along with cellulases and xylanases, further increased xylose yields by 12% and 7%, respectively.
Adding both these enzymes, along with core cellulases and xylanases, allowed the xylose yield
to reach as high as 74%. Hence, the removal of side chains which impede endoxylanase activity
could enhance xylose yields significantly [67, 74, 206]. However, the total enzyme loading
employed to obtain this conversion was 44 mg/g glucan, which is not viable for industrial
processing. Therefore, systematically screening the critical endoxylanases and determining the
minimum amounts of accessory enzymes is necessary to decrease the total enzymes loading
without sacrificing hydrolysis performance.

111

100%
Glucose
Xylose

Sugar Yiled

80%

60%

40%

20%

0%
Core

Core +
Xylananses

Core + LβX Core + LβX + Core + LβX + Core + LβX + Core + LβX +
Xylanases Xylanases + Xylanases + Xylanases +
LαGl + LArb
LαGl
LArb

Figure 6.2 Glucose (blue bar) and xylose (red bar) yield after 24 h hydrolysis of AFEX-treated corn stover for different
enzyme cocktails. Core enzymes only contain CBH I, CBH II, EG I (4 mg/g glucan each) and βG (2 mg/g glucan).
Endoxylanases include LX1, LX2, LX3, LX4, LX5 and LX6 (4 mg/g glucan each). Accessory hemicellulases LArb, LαGl and
LβX were loaded at 2 mg/g glucan each.

112

6.3.2

Screening endoxylanases

In Figure 6.2, all 6 xylanases were loaded together to evaluate their performance along with LβX,
LαGl and LArb and also to prevent the risk of missing any synergism among the xylanases.
However, not all xylanases may be necessary to bring about efficient xylan hydrolysis. In order
to determine the most important xylanases or their combinations, a two level (0 and 4 mg/g
glucan for low and high level of enzymes loading), six factor (LX 1 to LX 6) full factorial
experiment was carried out by fixing the remaining enzyme loading (CBH I, CBH II and EG I at
4 mg/g glucan each; βG, LβX, LαGl and LArb at 2 mg/g glucan each).

Figure 6.3 Glucose and xylose yield after 24 h hydrolysis of AFEX-treated corn stover for
different xylanase loadings. LX1 to LX6 were loaded as different combinations (from single
enzyme loading to 5 enzymes added simultaneously). Open symbol denotes no LX3 or LX4
were included in the mixture. Left half filled symbol denotes LX3 was included in the

113

mixture. Right half filled symbol denotes LX4 was included. Close symbol denotes both
LX3 and LX4 were present in the mixture.
Results of the 24 h hydrolysis results for AFEX corn stover are shown in Figure 6.3 which
depicts the glucose versus xylose yields. The xylose yield shows a strong linear relationship with
the glucose yield. This reconfirmed previous findings that high glucose yields can be achieved at
higher xylose yields for AFEX treated corn stover [198]. In this experiment, xylanase loading
increased depending on the number of xylanases involved in the mixture. Experiments
performed by adding individual xylanases showed that LX3 gave the highest conversion
followed by LX4. For binary LX mixtures, the best mixture contained both LX3 and LX 4.
Mixtures containing neither of these two enzymes resulted in 10% or more drop in both glucose
and xylose yield. Combinations containing either LX3 or LX4 exhibited moderate conversions.
For ternary mixtures, and other higher multiple LX loadings, LX3 and LX4 gave a similar trend
which suggests that both LX3 and LX4 are superior compared to other xylanases.
6.3.3

Optimum cellulase to xylanase ratio

Based on previous results, both LX3 and LX4 were chosen as supplementary xylanases for
further studies. As xylose and glucose yields are highly correlated, the ratio between cellulases
and xylanases loaded was further optimized.
In Figure 6.4, varying ratios of xylanases (LX3 and LX 4 at equi-mass loading) were
supplemented along with fungal core cellulases (CBH I, CBH II and EG I at equi-mass loading)
for a fixed total protein loading of 20 mg/g glucan. A fixed amount (2 mg/g glucan) of other
enzymes (βG, LβX, LaGl and LArb) were also loaded. Both the glucose and the xylose yields are
shown in Figure 6.4. Without any xylanases, the glucose and xylose yields dropped to 63% and
43%, respectively. As EG I has some xylanase activity [70, 71], even in the absence of any other

114

xylanases, a significant xylose yield was obtained by the endocellulase in the presence of LβX.
Supplementing the mixture with 5% xylanase increased both the glucose and xylose yields
significantly. At 25% xylanase supplementation, the highest observed glucose and xylose yields
were seen. When no cellulases were loaded, the glucose yield drops to less than 10%.
Interestingly, xylanase loadings, ranging from 15% to 75% of total enzyme added, gave
comparable glucose (>70%) and xylose yields (>60%).

100%

Sugar Yield

80%
60%
40%
Glucose

20%

Xylose
0%
0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Fraction of xylanases (LX3 and LX4) in cellulasexylanases mixture
Figure 6.4 Glucose (blue) and xylose (red) yield after 24 h hydrolysis of AFEX-treated corn
stover for different ratios of endoxylanases LX3 and LX4 in total enzymes (CBH I, CBH II,
EG I, LX3 and LX4) loading of 20 mg/g glucan. All enzymes were loaded at equi-mass ratio,
except βG, LβX, LαGl and LArb that were additionally supplemented at 2 mg/g glucan
each.

115

6.3.4

Synergism between endoxylanases (LX3 and LX4)

The optimal endoxylanase loading is one-third of the amount of cellulases added for hydrolysis.
It is interesting to compare the synergistic role of each xylanase in the mixture. Based on
previous results, cellulases (CBH I, CBH II and EG I) were loaded at 5 mg/g glucan each,
xylanases (LX3 and LX4) were loaded at 5 mg/g glucan total and accessory enzymes (βG, LβX,
LαGl and LArb) were loaded at 2 mg/g glucan each. Several reports highlight the synergism
between exo-glucanases and also between different families of hemicellulases [139, 207, 208]. It
is interesting to determine if there is similar synergism between the endoxylanases. Figure 6.5
shows that supplementing only LX4 or LX3 along with core cellulases and accessory enzymes
results in xylose yields of 60% and 65%, respectively. However, synergistic combinations of
LX3 and LX4 resulted in a greater than 70% xylose yield. In addition, the glucose yield also
benefit from including both LX3 and LX4. Table 6.1 shows that LX3 and LX4 belong to GH
family 10 and 11, respectively. These results suggest that both family 10 and 11 glycosyl
hydrolases are necessary to achieve complete xylan conversion for AFEX treated corn stover. It
is normally believed that family 10 xylanases are more versatile and efficient than family 11
xylanases [67, 207]. Family 10 xylanases cleave β-1,4 linkages from at least one unsubstituted
xylopyranosyl residue adjacent to substituted xylopyranolsyl residues towards the reducing end.
However, family 11 xylanases only cleave linkages adjacent to at least two unsubstituted
xylopyranosyl residues [67]. Our results are also consistent with Banerjee et al., who found that a
combination of GH family 10 and 11 fungal xylanases help increase both glucose and xylose
yields [74]. Ratios of LX3/(LX3+LX4) between 0.4-0.7 yielded comparable total
monosaccharides yields. These results reconfirm previous observations that both LX3 and LX4
are required in order to achieve higher monosaccharides yields.

116

95%
90%
85%
Sugar Yield

80%
75%
Glucose

70%

Xylose
65%
60%
55%
50%
0

0.2

0.4

0.6

0.8

1

Ratio of LX 3 in xylanases (LX3 + LX4)
Figure 6.5 Glucose (blue) and xylose (red) yield after 24 h hydrolysis of AFEX-treated corn
stover for different ratios of LX3 and LX4. 5 mg/glucan of each CBH I, CBH II, EG I and
(LX3 +LX4) were added to the hydrolysis. Other enzymes (βG, LβX, LαGl, and LArb)
were each added at 2 mg/g glucan.
6.3.5

Minimizing loading of accessory enzymes (LβX, LαGl and LArb)

In all of the above studies, LβX, LαGl and LArb loading was in excess. In order to determine the
minimum loading of each enzyme, different amounts of each were loaded while the other two
were kept in excess. Glucose yields for all experiments were comparable (data not shown) which
suggests that these enzymes do not influence glucan hydrolysis. However, there was a significant
change in the xylose yields as shown in Figure 6.6. The minimum loading for LβX, LαGl and
LArb was 0.6, 0.8 and 0.6 mg/g glucan, respectively. Note: LβX plays a very important role on
xylose yield. Without LβX, the xylose yield is very low, even when LαGl and LArb are loaded.

117

Xylose Yield

80%

60%

40%
LβX
LArb
LαGl

20%

0%
0

0.2

0.4

0.6

0.8

1

Enzymes loading (mg/g glucan)
Figure 6.6 Xylose yield after 24 h hydrolysis of AFEX-treated corn stover in presence of
accessory enzymes. LβX, LArb and LαGl were loaded individually at different protein
loadings, in presence of an excess of the other two enzymes (1 mg/g glucan each). Major
cellulases (CBH I, CBH II and EG I), hemicellulases (LX3 and LX4) were loaded at 5 and
2.5 mg/g glucan each, respectively. Additional βG (2 mg/g glucan) was added to prevent
cellobiose build-up.
6.3.6

Comparison of monosaccharides yield for optimized and crude commercial enzyme

preparations
The previous experiments helped define the optimal cellulase and hemicellulase loadings
necessary to maximize monomeric glucose and xylose yields from AFEX treated corn stover. It
was interesting to compare the hydrolytic activity of optimized cellulase-hemicellulase cocktail
with purified core cellulases and crude commercially available enzymes on pretreated biomass
with respect to the maximum monosaccharide yield achieved. in Figure 6.7.

118

From Figure 6.7, at a fixed total protein loading (30 mg/g of glucan), it is clear that the
commercial enzyme (Accellerase 1000) has limited hydrolytic activity on both glucan (70%) and
xylan (20%) fractions for AFEX treated corn stover due to the lack of suitable hemicellulase
activity. The core cellulase mixture (CBH I, CBH II and EG I) had slightly better glucan
conversions but lower xylan yields (<5%) compared to the commercial preparations.
Interestingly, the inclusion of xylanases and xylosidases enhanced both glucose and xylose
yields significantly. When small amounts of accessory hemicellulases (LαGl and LArb) were
included in the core cellulase-xylanase mixtures, glucose yields were unchanged while xylose
yields increased to >70%. For the total monosaccharide yield (Figure 6.7C), Accellerase 1000 at
30 mg/g glucan loading has a similar yield (50%) compared to the optimized cocktail at around 7
mg/g glucan.
In order to achieve monosaccharides yields greater than 80%, which is necessary for an
economical industrial process, the optimized cocktail concentration needed to be around 22 mg/g
glucan while the non-optimal cellulase-xylanase mixtures required more than 33 mg/g glucan.
These results demonstrate how trace amounts of important accessory hemicellulases can further
enhance the overall polysaccharide hydrolysis yields for AFEX pretreated corn stover. About 1.2
mg/g glucan total loading of LArb and LαGl can decrease the overall enzyme loading by over
33%. Furthermore, if the cocktail does not contain specific activities, such as β-xylosidase, αarabinofuranosidase and α-glucuronidase, no matter how much of the other enzymes are loaded,
it is impossible to achieve high xylose hydrolysis yields.

119

100%

A

90%

Glucose Yield

80%
70%
60%
Accellerase 1000

50%

Core cellulases

40%
30%

Core Cellulases + Xylanases

20%
Core Cellulases + Xylanases +
Accessory hemicellulases

10%
0%
0

10
20
30
Total enzymes loading (mg/g glucan)

40

Figure 6.7 Glucose (A), xylose (B) and total monosaccharide (C) yields after 24 h hydrolysis
of AFEX-treated corn stover for various enzyme cocktails. Accellerase 1000 is a
commercial preparation. Core cellulases contain CBH I, CBH II and EG I at equi-mass
loading along with 2 mg/g glucan βG. Xylanases (LX3 and LX4 at equi-mass ratio) to
cellulases (CBH I, CBH II and EG I) ratio is fixed at 1:3. LβX, LαGl and LArb were
loaded at 0.6, 0.8 and 0.6 mg/g glucan, respectively).

120

Figure Figure 6.7 (cont’d)

80%

B

70%

Xylose Yield

60%
50%
40%
30%
20%
10%
0%
0

10
20
30
Total enzymes loading (mg/g glucan)

40

10
20
30
Total enzymes loading (mg/g glucan)

40

90%

C

80%

Sugar Yield

70%
60%
50%
40%
30%
20%
10%
0%
0

121

6.4 Conclusion
In this work, an optimized cocktail of xylanases and accessory enzymes was identified for AFEX
treated corn stover. This cocktail included both fungal cellulases (CBH I, CBH II, EG I and βG)
and bacterial hemicellulases (LX3, LX4, LArb, LαGl and LβX). The optimized cocktail can
hydrolyze AFEX treated corn stover, resulting in glucose and xylose hydrolysis yields greater
than 80% and 70%, respectively, at a reasonable protein loading (~20 mg/g glucan). Adding
endoxylanases increases both xylose and glucose yields significantly (along with a suitable βxylosidase). Supplementation of accessory α-arabinofuranosidase (LArb) and α-glucuronidase
(LαGl) further increased xylose yields by 20 percentage points. This study clearly demonstrates
that for biomass pretreated by a specific technology (for example, AFEX or similar alkaline
pretreatments), it is both possible, and important, to tailor-make specific enzyme cocktails with
optimal individual enzyme ratios to achieve higher monosaccharide yields.
This study was carried out at low solids loading (0.2% glucan or 0.58% solids basis, respectively)
due to mass-transfer limitations typical for microplate based assays [177]. Therefore, the
monosaccharide yield and enzyme loadings discussed here are valid for the best case scenario
and there were no significant end-product inhibition or mass transfer limitations typically
encountered during high solid loading (15-30% solids loading)-based saccharification. These
results should mimic a simultaneous saccharification and fermentation process where the
monosaccharides are directly fermented to ethanol without allowing a significant build of sugars.
For an industrial high solid loading hydrolysis, more βG and βX might be necessary in order to
overcome the build-up of sugar oligomers due to the inhibition of enzymes at high
concentrations of monosaccharides and potential unproductive binding to lignin.

122

AFEX pretreated biomass, which contains nearly all of the original xylan content from untreated
biomass, is very different from acid pretreated biomass in terms of its physicochemical
composition and enzymatic digestibility [19, 209, 210]. The efficacy of any pretreatment is
normally evaluated by enzymatic hydrolysis at specific conditions (for example, enzyme loading,
hydrolysis time, solid loading). In order to achieve an unbiased comparison between pretreated
samples, enzymes used for evaluation should be able to reflect the true efficacy of the
pretreatment. When comparing AFEX versus dilute acid pretreated biomass, one should include
necessary hemicellulase and accessory enzyme activities in the enzymatic cocktail. Otherwise
the lack of specific activities could result in underestimating the true digestibility of AFEX
pretreated biomass. In addition, for other low severity pretreatments (such as alkaline peroxide)
which do not remove most of the hemicellulose fraction, the results in this paper could also be
useful for the development of more balanced enzyme cocktails in order to evaluate the
pretreatment effectiveness.

123

CHAPTER 7 INTERACTION OF TRICHODERMA REESEI CELLULASES WITH
UNTREATED, AFEX, AND DILUTE-ACID PRETREATED BIOMASS
7.1 Introduction
From previous chapters, enzyme cocktail can be optimized to improve hydrolysis yield. Among
those optimized cocktail, CBH I, CBH II and EG I are the three major enzymes contribute to >60%
of total enzyme loading. If one can improve their efficiency by understand the mechanism of
these enzymes during hdyrolsysi, a more efficient hydrolysis process can be expected.
All of the three enzymes have CBD. It has been suggested that almost all of the hydrolysis
parameters, such as temperature [156], pH [211], ionic strength [149], solids loading [93],
enzyme type [212], pretreatment and substrate ultrastructure [213] can influence cellulase
binding. The limitation of current enzyme binding studies are shown in Table 2.5.
Among those techniques, Fast Performance Liquid Chromatography (FPLC) based separation
and quantification of proteins is able to quantify protein based on the differences in their
isoelectric points (pI). Pretreated cell walls have additional UV-absorbing compounds (e.g.,
aldehydes, furans, phenolics) that can interfere with the separation and quantification of enzymes
using IEX (Figure 7.1) [19]. In order to overcome this problem and study the binding behavior of
purified cellulase (i.e., CBH I, CBH II and EG I) mixtures on untreated (UN-CS), dilute acid
(ACID-CS) and ammonia fiber expansion pretreated corn stover (AFEX-CS), we report here a
modified, semi-automated HT-FPLC method. This method utilizes gel filtration prior to ion
exchange based cellulase separation and quantification using a 96-well microplate assay format
that is compatible with a high-throughput hydrolytic assay method recently reported by our
group [71, 177]. Using this method we are able to accurately quantify unbound CBH I, CBH II

124

and EG I within untreated and pretreated corn stover hydrolyzates. This method was then used to
study multiple enzyme and pretreated biomass combinations to illustrate its utility. Our results
reveal the importance of how different pretreatments (i.e., AFEX and dilute-acid) can influence
accessibility and total available binding sites for individual cellulases as well as how these
enzymes can compete and/or cooperate with each other for those binding sites.

300

100
UV 280
80

B%
200

60
150
EG I

40

CBH II

100

CBH I

50

B%

UV 280 (mAU)

250

20
0

0
0

5

10

15

20

Elution (mL)
Figure 7.1 Separation of individual cellulases by anion exchange chromatography after 20fold dilution of AFEX corn stover hydrolyzate supernatant in pH 7.5, 20 mM Tris buffer
without removal of lignocellulose derived UV-absorbing decomposition products. Enzymes
were eluted by applying a linear gradient of 1M NaCl to the column (dotted line; shown
as %B on secondary Y-axis).
7.2 Materials and Methods
7.2.1

AFEX pretreatment

Detailed information is described in 4.2.1

125

7.2.2

Dilute acid pretreated corn stover

The dilute acid pretreatment was performed with a 1.0 L Parr reactor made of Hastelloy C (Parr
Instruments, Moline, IL, USA). CS was presoaked in 1.0% w/v dilute sulfuric acid solution at
5.0% solids (w/w) overnight. The total weight of the pretreatment mixture was 800 g. The
presoaked slurry was transferred into the reactor, which was then sealed and fitted to the impeller
driver motor which was set at 150 rpm. The vessel was lowered into a hot sand bath and heated
rapidly (within 2 min) to an internal temperature of 140 ± 2°C and maintained at 140 ± 2°C in
the fluidized heating sand bath for 40 min. At the end of the reaction time, the reactor was cooled
to below 50°C in a water bath. The dilute acid pretreated CS slurry was filtered through
Whatman no. 1 filter paper. Details of the apparatus, experimental procedure and combined
severity calculation are described elsewhere [214, 215]. After pretreatment, the dilute acid
treated corn stover (ACID-CS) solids were washed to neutral pH, dried at room temperature in a
o

fume hood, milled using a 0.1 mm screen (as described previously) and stored at 4 C. The
composition of the ACID-CS solids was approximately 60.6% glucan, 3.3% xylan, and 32.9%
lignin.
7.2.3

Enzyme purification

Detailed information is given in Chapter 3 and in a paper published by Gao et al[71, 198].

7.2.4

Cellulase Binding and Quantitation

For enzyme quantification study, the total enzyme loaded was assumed to be equal to the enzyme
bound to the solid biomass plus the free enzyme left in the supernatant. Thus, by assaying the
amount of free enzyme in the hydrolyzate, one can determine the amount of enzyme bound to the

126

biomass. An illustrative schematic highlighting the cellulase adsorption and HT-FPLC based
quantification methodology is shown in Figure 7.2. A 2.2 ml deep-well microplate (Lot # 780271,
Greiner, Monroe, NC) was used to load 200 µl of 1.45% (w/v) AFEX-CS/UN-CS or 200 ul of
0.825% (w/v) ACID-CS along with 25 µl 1M citrate buffer (pH 4.8) and 275 µl of enzyme
mixtures using epMotion 5070 automatic pipetting work station (Eppendorf, Hamburg, Germany)
as described previously [177]. Each well contained 50 mM citrate buffer and 1 mg glucan
o

equivalent solids in a total reaction volume of 0.5 ml. After incubation for 2 hours at 4 C at 250
rpm, the supernatant was separated from the insoluble solids by filtering through a 0.45 µm low
protein binding hydrophilic microplate based filter (Lot # R6PN00144, Millipore, Ireland). The
FPLC system (ÄKTA purifier) and autosampler (A905) used for enzyme filtrate separation and
quantification were from GE Healthcare (Buckinghamshire, United Kingdom). The enzyme
filtrate was loaded into the FPLC autosampler for gel filtration chromatography to isolate the
cellulases from the UV-absorbing background (Figure 7.2). The gel filtration column was packed
with 28 mL of Superdex 200 (Lot #S6782, Sigma-Aldrich, St. Louis, MO) media in a XK 16/20
column (Lot #18-8773-01, GE Healthcare). While during eluting out 2 column volumes (CV) of
20 mM Tris buffer (pH 7.5), the enzyme rich fractions were collected as 6 mL aliquots. Part of
the aliquots (0.5 mL) were then injected into a Mono Q column (Lot #17-5179-01, GE
Healthcare) followed by 6 CV linear gradient elution with 1M NaCl in 20 mM pH 7.5 Tris buffer.
The concentration of individual enzymes was correlated to the elution peak area detected at UV
280 nm and calculated using the Unicorn 5.11 software. The binding studies are performed at 4
o

C at different individual cellulase loading ranging from 25 to 450 mg/g glucan. CBH I, CBH II

and EG I bindings were studied individually as single enzyme systems. For binary enzyme
mixtures, pairs of CBH I/CBH II, CBH I/EG I and CBH II/EG I were loaded at equal mass

127

loading (e.g., 25 mg/g cellulase loading binary mixture experiment comprised of 25 mg/g glucan
loading of enzyme A and B each, respectively. Hence the total cellulase loading was actually 50
mg/g glucan for this binary mixture experiment.), and each enzyme’s binding characteristics
were studied in the presence of the other enzyme present in the pair. Similarly, for ternary
enzymes system, equal mass ratios of all three enzymes were loaded and each individual
enzyme’s binding characteristics were studied.

128

Binding studies are performed
in the deep well microplate.
Incubation at 4ºC for 2 hours.

Reaction plate
Hydrolyzate was filtered through
low protein binding filter plate.

Filter plate

Receiver plate
Hydrolyzate was injected in
Superdex 200 gel filtration column.

Figure 7.2 Two-step high-throughput HT-FPLC method for separation and quantification of complex cellulase mixtures
present in lignocellulosic biomass derived hydrolyzates. High molecular weight (>50 kDa) cellulases are separated from AFEX
pretreated corn stover hydrolyzate derived UV-absorbing components (e.g., phenolics) by Superdex 75 gel filtration and then
quantified by anion exchange chromatography (IEX). Enzymes were eluted applying a linear gradient of 1M NaCl (dotted line;
shown as %B on secondary Y axis).

129

Figure 7.2(cont’d)

UV 280 (mAu)

400
300
200

Cellulase

100
0
0

20

40

60

Elution (mL)

UV 280

100
CBH II

%B

150

80
60

100

EG I
40

CBH I

50

20

0

0
0

5

130

10
15
Elution (mL)

20

%B

UV 280 (mAU)

200

7.2.5

Langmuir Model Fitting

The binding isotherms were fit to a Langmuir isotherm model using Matlab 7.0 (MathWorks Inc.,
Natick, MA). The curve fitting toolbox in Matlab was set using the Trust-Region algorithm to fit
the isotherm data. The Langmuir model is described using the following equation:

‫ܧ‬௕ ൌ

‫ܧ‬௕௠ ‫ܭ‬௔ ‫ܧ‬௙
1 ൅ ‫ܭ‬௔ ‫ܧ‬௙

Where Eb is the bound enzyme (mg/g glucan); Ebm is the total available binding sites (mg/g
glucan); Ef is the concentration of free enzyme present in liquid phase (mg/L) and Ka is the
association constant (L/mg).
7.3 Results and Discussion
7.3.1

FPLC Quantification

However, for real lignocellulosic biomass, directly injecting the lignocellulosic hydrolyzate
(even after 20 fold dilution) into the ion-exchange column results in poor protein binding to the
column and causes severe base line fluctuation during separation (Figure 7.1). CBH II cannot
bind to the column effectively and the baseline fluctuates considerably during the separation,
making it difficult to achieve reliable peak area quantification. Hence, desalting and buffer
exchange of the hydrolyzate using an appropriate buffer is necessary prior to IEX.
Thus, a preliminary gel filtration chromatography step was used to separate the high molecular
weight cellulases from the lower molecular weight background components. Large molecules,
such as enzymes, elute from the gel filtration column earlier than low molecular weight

131

compounds. The gel filtration step separated cellulases (CBH I, CBH II and EG I) from other cell
wall derived compounds that have significant UV absorption (Figure 7.2). In addition, gel
filtration acted as a buffer exchange step which is also necessary for subsequent ion-exchange
chromatographic separation (Note: enzymatic hydrolysis is done typically at pH 4.5-5.0 while,
the optimal ion-exchange separation takes place at pH 7.5 for T. reesei cellulases.) This pH
adjustment during gel-filtration depends on the exact protein pI and ion-exchange method
employed and can be optimized for other non-trichoderma enzymes as well.). After gel filtration,
the enzyme fractions were injected into an anion exchange column (Mono Q), and the bound
enzymes (CBH I, CBH II and EG I) were eluted using a linear gradient of NaCl (Figure 7.2).
Based on the differences in pI, CBH II (pI ~ 6.8) eluted out first, followed by EG I (pI ~ 4.5) and
CBH I (pI ~ 4.2). CBH I, CBH II and EG I were separated with good baseline resolution.
Calibration curves based on peak areas were used to determine the concentration of individual
enzymes. Cellulase mixtures of known concentration for individual enzymes that could be
reliably quantified by this method lies within the range of 50 µg/ml to 0.9 mg/ml (this
corresponds to 25 to 450 mg/g glucan loading). Linear regression standard curves with
2

satisfactory linearity (R >0.999) were obtained to determine unknown protein concentrations.
All standard enzymes mixtures of known concentrations had also undergone the exact same
sample preparation procedure as the enzymes in the biomass hydrolyzates to minimize the
potential error associated (e.g., non-specific binding of enzymes to microplate walls, enzyme loss
during microplate filtration and FPLC based gel filtration) with the absolute quantification. The
UV-900 multi-wavelength UV sensor in the current FPLC system is incorporated with a xenon
flash lamp which supplies a higher sensitivity and higher signal to noise ratio compared to
regular mercury lamps. To evaluate how UV signal drifts during long run time measurements

132

affects the reproducibility of this method, 98 samples of hydrolyzate containing around 0.3
mg/ml each of three cellulases were tested continuously for 3 days. The coefficients of variation
(based on detected protein peak areas) for CBH I, CBH II and EG I are 0.9%, 1.5% and 1.6%
respectively and there is no obvious increasing or decreasing trend of UV signal strength during
measurement. These results demonstrate that the current FPLC methodology is both robust and
reproducible.
One could speculate that inclusion of an enzyme blank (e.g., hot water extract from pretreated
biomass alone) to re-calibrate the standard curve could overcome the lignin interference to UV
absorbance. However, this could be challenging because during the course of enzymatic
hydrolysis increasing amounts of soluble lignin are expected to be released [216]. Hence, the
standard curve would need to be adjusted for each time point sampled. Incorporating a gel
filtration step to separate the lignin (and other UV absorbing cell wall components) from the
enzymes in varying time-course hydrolyzates prevented such difficulty with satisfactory
reliability (data not shown). Another solution to this problem was illustrated by a recent study
that used a dual-wavelength spectral correction method to minimize lignin interference on UVVis spectrophotometric measurements to quantify cellulase adsorption to lignocellulosic biomass
[217]. Compared to other reported methodologies (Table 2.5) [79, 156], the current FPLC
method has certain advantages. First, the method gives reproducible and accurate results due to
semi-automation of the protocol (e.g., via use of a FPLC microplate based autosampler). Second,
by removing 280 nm UV-light absorbing background compounds using a gel filtration column
this method can be used as a tool to probe the binding properties of cellulases on real
lignocellulosic biomass (unlike previous binding studies that used purified cellulosic materials
devoid of these background compounds). Third, all major cellulases involved in hydrolysis

133

(CBH I, CBH II and EG I make up nearly 80% of total enzyme present in crude cellulase broths)
[71, 210] can be simultaneously monitored, allowing us to study protein-protein interactions for
real lignocellulosic biomass.
7.3.2

Binding isotherms for single, binary and ternary cellulase mixtures

Compared to UN-CS, both AFEX-CS and ACID-CS have much higher cellulase binding
capacities (Figure 7.3, Figure 7.4 and Figure 7.5). For example, when CBH I was added alone at
high enzyme loading (450 mg/g glucan), only 60 mg/g glucan CBH I bound to UN-CS whereas
190 and 147 mg/g glucan CBH I was bound to AFEX-CS and ACID-CS, respectively. This
suggests that both pretreatments significantly increase cellulase accessibility for corn stover.
These results agree with earlier studies by Jeoh et al. [33] and Kumar et al. [153, 218] showing
that pretreated corn stover has higher CBH I binding capacity. We also found similar results for
CBH II and EG I.

134

200

A

Bound CBH I (mg /g glucan)

180
160
140
120
100
80
60

CBH I
CBH I with CBH II
CBH I with EG I
CBH I with CBH II and EG I

40
20
0
0

100

200

300 400 500 600
Free CBH I (mg/L)

700

800

900

Figure 7.3 Binding isotherms for CBH I (A), CBH II (B) and EG I (C) on AFEX pretreated
corn stover. The extent of binding for individual enzymes was measured for single, binary
and ternary cellulase mixtures.

135

Figure 7.3 (cont’d)

200

CBH II
CBH II with CBH I
CBH II with EG I
CBH II with CBH I and EG I

Bound CBH II (mg /g glucan)

180
160

B

140
120
100
80
60
40
20
0
0

100

200

300 400 500 600
Free CBH II (mg/L)

700

800

900

400
EG I
C
EG I with CBH I
EG I with CBH II
EG I with CBH I and CBH II

Bound EG I (mg /g glucan)

350
300
250
200
150
100
50
0
0

100

200

300 400 500 600
Free EG I (mg/L)

136

700

800

900

200

CBH I
CBH I with CBH II
CBH I with EG I
CBH I with CBH II and EG I

Bound CBH I (mg /g glucan)

180
160

A

140
120
100
80
60
40
20
0
0

100

200

300 400 500 600
Free CBH I (mg/L)

700

800

900

Figure 7.4 Binding isotherms for CBH I (A), CBH II (B) and EG I (C) on dilute acid
pretreated corn stover. The extent of binding for individual enzymes was measured for
single, binary and ternary cellulase mixtures.

137

Figure 7.4 (cont’d)

200

CBH II
CBH II with CBH I
CBH II with EG I
CBH II with CBH I and EG I

Bound CBH II (mg /g glucan)

180
160
140

B

120
100
80
60
40
20
0
0
400

200

300 400 500 600
Free CBH II (mg/L)

700

EG I
EG I with CBH I
EG I with CBH II
EG I with CBH I and CBH II

350
Bound EG I (mg /g glucan)

100

300

800

900

C

250
200
150
100
50
0
0

100

200

300 400 500 600
Free EG I (mg/L)

138

700

800

900

200
Bound CBH I (mg /g glucan)

180

A

CBH I
CBH I with CBH II
CBH I with EG I
CBH I with CBH II and EG I

160
140
120
100
80
60
40
20
0
0

100

200

300 400 500 600
Free CBH I (mg/L)

700

800

900

Figure 7.5 Binding isotherms for CBH I (A), CBH II (B) and EG I (C) on untreated corn
stover. The extent of binding for individual enzymes was measured for single, binary and
ternary cellulase mixtures.

139

Figure 7.5 (cont’d)

200
Bound CBH II (mg /g glucan)

180

B

CBH II
CBH II with CBH I
CBH II with EG I
CBH II with CBH I and EG I

160
140
120
100
80
60
40
20
0
0

100

200

300 400 500 600
Free CBH II (mg/L)

700

800

900

400
Bound EG I (mg /g glucan)

350

C

EG I
EG I with CBH I
EG I with CBH II
EG I with CBH I and CBH II

300
250
200
150
100
50
0
0

100

200

300

400 500 600
Free EG I (mg/L)

140

700

800

900

Although the binding isotherms and overall binding capacities for individual enzymes are quite
different for AFEX-CS, ACID-CS and UN-CS, the relative order of binding capacities among
the samples is the same for each protein. At 450 mg/g glucan protein loading EG I added alone
had the highest binding capacity (374, 160 and 103 mg/g glucan for AFEX-CS, ACID-CS and
UN-CS, respectively), followed by CBH I (190, 147 and 60 mg/g glucan for AFEX-CS, ACIDCS and UN-CS, respectively) and CBH II (154, 97 and 29 mg/g glucan for AFEX-CS, ACID-CS
and UN-CS, respectively).
We studied the binding isotherms for binary mixtures of enzymes in which one enzyme’s
binding properties were investigated in the presence of another enzyme at equivalent protein
mass loading. Overall, these isotherms demonstrate that the relative extent of binding depends on
both the type of cellulase and the substrate. For AFEX-CS, two different types of binding
phenomena were observed; one was cooperative binding for CBHs in the presence of EG I, the
other was competitive binding between CBH I and CBH II. For CBH I-CBH II binary mixtures,
the presence of either exocellulase prevented the other enzyme from binding and resulted in
reduced levels of bound enzymes. For CBH I-EG I and CBH II-EG I mixtures, presence of EG I
significantly enhanced CBH I and CBH II binding to AFEX-CS. At 100 mg/g glucan enzyme
loading for each component, EG I increased CBH I and CBH II binding to AFEX-CS by 71%
and 42%, respectively, compared to the case in which CBH I or CBH II were added alone. This
enhancement in exocellulase binding due to EG I was not observed when individual enzyme
loadings exceeded 200 mg/g glucan. Under those conditions, the binding capacities for CBH I or
CBH II (each in the presence of EG I) were significantly lower compared to each exocellulase
added alone. Previous saccharification experiments for AFEX-CS have shown significant
synergism between CBH I-EG I and CBH II-EG I mixtures hydrolyzing both the glucan and

141

xylan fractions of the substrate compared to CBH I-CBH II combinations [71]. This hydrolytic
synergism between exo- and endo- cellulases on AFEX-CS is consistent with both CBH II and
CBH I exhibiting cooperative binding in presence of EG I. On the other hand, no cooperative
binding occurred between any of the binary mixtures for UN-CS; only competitive binding was
observed. For ACID-CS also, only competitive binding was observed. Introduction of another
enzyme to the binary mixture was found to always decrease the binding capacity of the other
enzyme in the pair.
In addition, CBH I was found to compete more strongly than CBH II for available binding sites
in the presence of EG I. Since though both cellulases have highly homologous CBD domains
their binding affinity is considerably different, suggesting the CD would also play an important
role of directing exo-cellulase binding to crystalline cellulose as suggested elsewhere as well
[210]. CBH I is a processive enzyme that moves along the cellulose chain during hydrolysis [57].
For complete biomass hydrolysis, it is necessary for EG I, which creates endo-cuts on the
cellulose chain, to return to the liquid phase when competing with CBH and hence bind on
another cellulose chain to create additional endo-cuts. Other binding properties of binary enzyme
mixtures have been reported on isolated crystalline cellulose. Jeoh et al. found cooperative
binding for Cel5A and Cel6B from T. fusca on bacterial microcrystalline cellulose at 50 °C while
competitive binding was observed at 5°C [147]. Medve et al. found no cooperative binding
between CBH I and EG II on Avicel at 4 °C [79]. However, others have shown competition
during binding at 4°C on Avicel for CBH I and CBH II [156, 219].
For ternary enzyme mixtures with AFEX-CS as substrate, CBH I binding reflects both
cooperative and competitive effects depending on the total enzyme loading employed. EG I
increased the binding of both exocellulases at lower enzyme loading (<150 mg/g glucan for CBH

142

I and <100 mg/g glucan for CBH II). On the other hand, CBH I and CBH II compete with each
other and gave lower maximum binding capacities than previous single and binary component
mixtures. EG I was unable to bind cooperatively with either CBH I or CBH II, reducing the
extent of bound EG I dramatically. As the enzyme loading increased, these trends become more
obvious. At lower protein loadings, the available AFEX-CS substrate binding sites are
sufficiently numerous to allow cooperative binding between enzymes. But as the protein
loadings increase, fewer binding sites are available for all enzymes resulting in increased
competitive binding. CBH I had the highest binding affinity followed by EG I and CBH II for the
ternary system. Similar results were found for ACID-CS, with CBH I having the highest binding
capacity, followed by EG I and CBH II.
To assist the reader in understanding these complex binding patterns, we have summarized these
results in a tabular form. In Table 7.1, the cooperative and competitive binding in binary enzyme
mixture for different substrates was shown to vary with the total enzyme loading employed. It is
also interesting to note, for some enzymes mixtures such as CBH I and CBH II in AFEX-CS,
neither cooperative nor competitive binding was observed at low enzyme loading, which
suggests independent binding of the enzyme pair on biomass.

143

Table 7.1 Cooperative and competitive binding among various binary cellulase mixtures for AFEX pretreated (AFEX-CS),
dilute-acid pretreated (ACID-CS) and untreated (UN-CS) corn stover. Binding types (cooperative versus competitive) are
generalized in the table and the respective enzyme loading ranges within which that particular binding behavior is seen are
shown in brackets.
Binding type and range (mg/g glucan)
Bound

CBH I

Enzyme
In presence of

Substrates

AFEX-CS

ACID-CS

UN-CS

CBH II

EG I

CBH II

EG I

CBH I

EG I

CBH I

CBH II

0 (25~150)

+ (25~200)

0 (25~50)

+ (25~200)

0 (25~50)

0 (25~50)

- (150~450) - (200~450)

-(150~450)

- (200~450)

- (50~450)

- (50~450)

0 (25~50)

0 (25~50)

0 (25)

0 (25)

0 (25)

0 (25~50)

- (50~450)

- (50~450)

- (25~450)

- (25~450)

- (25~450)

- (50~450)

0 (25~50)

0 (25~50)

- (50~450)

- (50~450)

- (25~450)

- (50~450)

0 (25~50)
- (25~450)

+ : Significant cooperative binding observed.
-: Significant competitive binding observed.
0: No significant influence on binding in presence of background enzyme.

144

- (50~450)

Table 7.2 Langmuir type (single-site model) binding isotherm parameters for individual cellulase enzymes binding to
untreated (UN-CS), ammonia fiber expansion (AFEX-CS) and dilute acid treated (ACID-CS) corn stover for various single,
binary and ternary enzyme mixtures.
AFEX-CS
ACID-CS
UN-CS
Kax10

Ebm
mg/g

3

CBH I

CBH II

EG I

Ebm
mg/g

3

Kax10

L/mg
1.5
3.4
103.5*

glucan
426.6
204.4
104.2

104.4*
3.1
7.7
123.9*

76
233.5
110
103.6

26.1
80.3
37.8
35.6

0.93
0.95
0.85
0.9

130.9
95.9
129.1
72.3

61.7
88.3
54.3
72.9

37.4
53.5
32.9
44.2

0.91
0.94
0.94
0.89

10.3
91.4
175.8
102.8

29.9
25.4
14.5
21.7

10.3
8.7
5.0
7.5

0.99
0.74
0.67
0.5

292.9*
19.8
35.3
15.1

45
500.4
159
282.1

15.5
172.1
54.7
97.0

0.59
0.99
0.94
0.94

104.1
23.4
36.9
15.6

36.5
166
76.7
128.7

22.1
100.6
46.5
78.0

0.57
0.99
0.97
0.97

37.2
27.5
10.9
29

23
97
62.2
53.6

7.9
33.4
21.4
18.4

0.82
0.84
0.8
0.85

85.7

92.6

31.9

0.87

80.4

39.7

24.1

0.89

39.7

35.3

12.1

0.64

2

R
0.99
0.98
0.98

L/mg
246.5
227.4
184.4

glucan
133.7
83.5
83

bioma
ss
81.0
50.6
50.3

R
0.94
0.96
0.95

Ebm
mg/g

3

biomas
s
146.8
70.3
35.8

Enzyme
Mixture
Alone
+ CBH II
+ EG I
+ CBH II +
EG I
Alone
+ CBH I
+ EG I
+ CBH I +
EG I
Alone
+ CBH I
+ CBH II
+ CBH I +
CBH II

Kax10

2

L/mg
2
7.6
14

biomas
glucan s
92.1
31.7
27.4
9.4
28.5
9.8

R
0.95
0.95
0.7

2

*

Cooperative binding was observed. Ka increased significantly in the presence of EG I for AFEX-CS.

#

This value was calculated based on the fitted isotherm Ebm (mg/g glucan) value and original glucan content (dry weight basis) for

each substrate.

145

7.3.3

Modeling Enzyme Binding Properties

Langmuir single-site adsorption model was used to describe cellulase binding to untreated and
pretreated corn stover under different enzyme loading conditions (single, binary or ternary
2

mixtures). The Ka, Ebm and coefficient of determination (R ) values are shown in Table 7.2. The
Ebm values based on both glucan and total biomass basis are shown for comparison. The model
2

fits for pretreated biomass, both AFEX-CS and ACID-CS, are much better (R >0.9) than those
for untreated corn stover, UN-CS. The Ebm for AFEX-CS and ACID-CS are much higher
compared to UN-CS, which is consistent with increased cellulose accessibility after pretreatment.
Hong et al. also found a linear relationship between increased substrate digestibility and
available binding sites for CBM on different cellulosic substrates [220]. Interestingly, Ebm for
AFEX-CS is typically higher than ACID-CS for these enzymes. Since the total biomass loading
for AFEX-CS is higher than ACID-CS (all experiments are based on equivalent glucan loading,
thus AFEX-CS has much higher xylan content than ACID-CS), one would expect the higher
xylan content for AFEX-CS could result in greater cellulase binding than ACID-CS. The effect
of individual cell wall components (e.g., xylan, lignin) on non-productive cellulase binding is not
clear based on these experiments and requires further investigation.
In addition, comparing the Ebm for CBH I, CBH II and EG I among different substrates suggest
that binding strengths for each enzyme depend on the substrate and cellulase type. For AFEX-CS
treated corn stover, EG I had the highest Ebm (500.4 mg/g glucan) followed by CBH I (426.6
mg/g glucan). For UN-CS, EG I and CBH I have a comparable Ebm (~95 mg/g glucan).

146

Interestingly, CBH II had the lowest Ebm (25.4 mg/g glucan) for untreated corn stover compared
to other cellulases. However, after pretreatment the Ebm for CBH II increased dramatically to
233.5 (AFEX-CS) and 88.3 (ACID-CS) mg/g glucan, respectively. These results suggest that the
preferred binding sites for CBH II, possibly inclusive of cellulose non-reducing ends [221], are
exposed and become more readily accessible after thermochemical pretreatments. As explained
previously, adding EG I increased CBH I and CBH II binding for AFEX-CS. This incremental
increase in binding was also reflected in the Ka values. The Ka value reflects the initial slope of
the isotherm, and hence corresponds to enzyme affinity for the substrate when the substrate is
present in excess. During cooperative binding of cellulases to AFEX-CS, the Ka values for CBH
increased dramatically for binary mixtures incorporating EG I compared to single exocellulases
which suggests that the CBH binding affinity to AFEX-CS increased significantly in the
presence of EG I. For ternary mixtures, Ka was also significantly higher than for single enzyme
experiments. For ACID-CS, Ka values (i.e., only for exocellulases in absence of EG I) are much
higher than AFEX-CS which suggests that removal of xylan can significantly modify the
substrate and hence alter its cellulase binding properties. In subsequent work, we hope to
explore these trends in the presence of xylanases, which may significantly alter the observed
binding patterns by exposing additional cellulose, perhaps in ACID-CS, but certainly in AFEXCS. In this work, the binding of enzymes onto the whole biomass was observed. However, it is
difficult to evaluate the individual contribution to binding for each cell wall component (e.g.,
crystalline cellulose, xylan, lignin, amorphous cellulose). Yang and Wyman added bovine serum
albumin as a “lignin blocker” to minimize unproductive binding during enzymatic hydrolysis of

147

dilute acid and AFEX treated corn stover [213]. They found that this approach benefitted
hydrolysis of AFEX-CS much less than it did for ACID-CS. This would lead one to speculate
that the lignin exposed during AFEX has lesser binding capacity for cellulases, possibly due to
lower lignin hydrophobicity as suggested recently [209], than lignin exposed during dilute acid
treatment [213]. Preliminary unpublished data from our lab also validates this finding. Though
binding of individual cellulase enzymes to lignin and hemicellulose fractions was not measured
in this study, it will be explored in future work.
7.3.4

Impact of AFEX pretreatment on cooperative cellulase binding

AFEX is a “dry to dry” (i.e., no separate liquid fraction arises from pretreatment) process and
the composition of pretreated biomass is almost identical to that of untreated biomass [15]. In
contrast, dilute acid pretreatment can remove a significant amount of hemicellulose and some
lignin [14]. Cellulose degree of polymerization (DP) is largely unchanged by AFEX (~ 60007000 for corn stover), unlike dilute acid treatment that results in decreasing the DP by 60-70%
[110]. Both of these thermochemical pretreatments are believed to increase cellulase accessibility
to cellulose (and hemicellulose, lignin) through various ultra-structural and physicochemical
changes that are being unraveled in recent years [14, 19, 111, 209].
One possible reason for cooperative binding between CBH-EG I binary mixtures for AFEX-CS
might be due to the ultrastructure of AFEX-CS compared to UN-CS and ACID-CS. AFEX has
been recently shown to create internal porosity within the cell walls by physically delocalizing
some of the hemicellulose and lignin to the outer wall surfaces. However, there is likely a
significant amount of xylan strongly associated with cellulose microfibrils after pretreatment
[209]. In contrast, acidic treatments hydrolyze and strip out most of the hemicellulose and some
of the lignin into a separate liquid stream during pretreatment [110, 210]. EG I is known to have

148

significant xylanase and xyloglucanase activity [70, 71], and hence could have prevented nonproductive binding of exo-cellulases to the hemicellulose fraction by binding to it instead.
Although EG I’s hydrolytic activity is mostly suppressed at 4ºC, it had marginal activity on
xylan tightly associated to cellulose especially at higher enzyme loadings (Note: EG I loading at
100 mg/g glucan or more resulted in ~2% xylan hydrolysis to soluble sugars at 4ºC). Removal of
this xylan sheath by EG I could have exposed the underlying cellulose surface, hence enhancing
exocellulase binding to cellulose. This also explains why at high total enzyme loading CBH
enzyme binding is much less when it is part of binary mixtures than when it is added as a single
enzyme. Since there are only trace amounts of hemicellulose left behind in the cell wall after
dilute acid treatment this cooperative binding phenomena is not readily observed for ACID-CS
(at least in presence of EG I). On the other hand, this phenomenon is also not observed for UNCS due to limited enzyme accessibility to the embedded cellulose sheathed by tightly crosslinked lignin and hemicellulose [33, 209].
7.4 Conclusions
Both AFEX and dilute acid pretreatment were found to increase cellulase binding to embedded
cellulose microfibrils within cell walls. Presence of EG I enhanced exo-cellulase cooperative
binding to AFEX pretreated cell walls likely due to presence of residual hemicellulose that
sheathed cellulose fibrils unlike what was seen for dilute acid treated cell walls. Competitive
binding among enzymes was also observed for certain substrates, cellulase combinations and
protein loadings employed. Though it was not possible to determine extent of non-productive
binding of cellulases from the current set of experiments, future studies with isolated lignin and
hemicellulose fractions will help gain a better understanding of this phenomenon. These studies
could assist in enzyme engineering efforts to minimize unproductive binding and help design

149

improved pretreatments that facilitate productive binding hence lowering the enzyme dosage
necessary for cost-effective enzymatic hydrolysis of lignocellulosic biomass.

150

CHAPTER 8 CELLULOSE CRYSTALLINITY AND LIGNIN: THEIR IMPACT ON
EFFICIENT CELLULASE ACTION
8.1 Introduction
In the previous chapter, the binding studies were done at 4 ºC on pretreated/untreated biomass.
Biomass is heterogeneous and 4 ºC inhibit enzyme activities significantly. To have a better
understand of how enzyme work, time course hydrolysis studies with tracking of individual
enzyme’s distribution between liquid phase and solid phase are indispensible. Both pure
cellulosic and lignocellulosic substrates are used in this chapter.
The extent of cellulase binding to crystalline cellulose has been generally found to be directly
correlated to the enzymatic hydrolysis rate [222-224]. However, the role of productive and nonproductive binding is difficult to discern from most experiments. Also, the role of cellulose
allomorph type on cellulase binding has not been studied before. Cellulose allomorph type (or
the lack of crystallinity) has been suggested recently to determine how difficult it is for fungal
cellulases to abstract individual glucan strands from the crystal surface prior to hydrolysis of the
glycosidic bond [210]. This suggests that different allomorph types may result in different
binding behavior for cellulases.
Lignin is thought to impede efficient enzymatic hydrolysis possibly due to unproductive binding
of cellulases to it and/or through steric hindrance facilitated by the lignin-carbohydrate complex
that decrease cellulose accessibility [33]. In addition, lignin derived degradation products
produced during pretreatment could further inhibit enzyme activity [20, 209]. Recalcitrance
towards enzymatic digestion is believed to be directly proportional to lignin content [102].
Delignification of biomass during or after pretreatment can enhance hydrolysis rate and overall

151

sugar yield [225]. Several studies have reported the beneficial effect of surfactants and other
proteins that can prevent unproductive binding of cellulases to lignin to some extent [145, 213,
226]. However, the affinity of lignin towards individual cellulases is still far from clear due to
the lack of reliable techniques to track individual enzyme binding present within complex protein
mixtures during saccharification. Doping extracted lignin to pure cellulosic substrates to mimic
pretreated biomass is one way to quantify the inhibitory role of lignin [115, 227]. However, the
major limitation of this approach is that lignin structure can be chemically or physically modified
during extraction and hence its affinity to enzymes could change. On the other hand, the
pretreated cell wall microstructure (e.g., porosity, lignin redistribution due to pretreatment) and
chemical linkages between different components (i.e., cellulose, hemicellulose and lignin) cannot
be simulated by simply recombining the respective purified components. Tracking enzyme
binding during hydrolysis has been carried out by measuring total crude protein concentration
and/or activities of unbound enzymes in the hydrolyzate [146, 153]. However, these methods
have limitations because it is difficult to differentiate individual enzyme binding from these
experiments.
To achieve a comprehensive understanding of cellulase binding to cellulosic biomass during
course of hydrolysis, we have applied a novel FPLC based methodology to quantify CBH I,
CBH II and EG I concentration in complex hydrolyzate mixtures [228]. Pure cellulose with
different crystal structures were included as well: microcrystalline cellulose I, cellulose II,
cellulose III, and regenerated amorphous cellulose. Lignocellulosic biomass materials included
in this study were: pretreated (ammonia fiber expansion or AFEX, dilute acid and ionic liquid
pretreated) corn stover to compare cellulase adsorption characteristics and overall digestibility as
a function of pretreatment type. These studies have revealed several interesting phenomena that

152

were previously unknown. These include; (i) relative cellulase binding affinity is different for
cellulose allomorphs and lignin-rich pretreated biomass, (ii) extent of unproductive cellulase
binding to lignin depends on pretreatment type and was found to negatively correlate with
hydrolysis yields, (iii) increased cellulase affinity for crystalline cellulose does not always
correlate with enhanced digestibility, and (iv) the relative thermostability of Trichoderma reesei
CBH II during course of saccharification is lower. We believe these findings will aid in
engineering low unproductive binding cellulases for novel pretreatments and lead to
development of economic enzyme recycling options.
8.2 Materials and Methods
8.2.1

Cellulosic substrates

Avicel (PH 101, Sigma-Aldrich, St Louis) was the pure cellulose source used to prepare all other
allomorphs. Cellulose III was prepared by soaking Avicel in anhydrous liquid ammonia at 100
o

C for 30 min. The samples were dried under nitrogen and purged overnight to remove residual
o

ammonia. Cellulose II was prepared by adding 25% NaOH to Avicel at 4 C for 60 min. The
slurry was then centrifuged, filtered and washed with water till neutral pH. Regenerated
o

amorphous cellulose (AC) from Avicel was prepared using 83% phosphoric acid at 4 C for 60
min based on published protocol [229] Cellulose II and AC were lyophilized to dryness prior to
o

storage at 4 C for future experiments. Cellulose crystallinity index was estimated using XRD
amorphous subtraction method [230].
8.2.2

AFEX pretreated corn stover

Detailed information is described in 4.2.1

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8.2.3

Dilute acid pretreated corn stover

Detailed information is described in 7.2.2
8.2.4

Ionic liquid pretreated corn stover

Ionic liquid pretreated corn stover was a generous gift from JBEI. Details on ionic liquid
pretreatment methodology are provided elsewhere [105].
8.2.5

Cellulase Purification

Detailed information is described in Chapter 3.
8.2.6

Enzymatic Hydrolysis

All hydrolysis experiments were performed in a 2.2 ml deep-well microplate (Lot # 780271,
Greiner, Monroe, NC) at 1% (w/w) glucan loading along with 50 mM pH 4.5 citrate buffer in
total reaction volume of 500 µl. 15 mg/g glucan (corresponding to 0.15 mg/mL) each of CBH I,
CBH II and EG I are loaded along with 2 mg/g glucan of βG (C Cocktail). This enzyme loading
allowed maximum digestion to be achieved for all cellulosic allomorphs within 48 h. For
pretreated biomass, the C Cocktail included an additional xylanase (5 mg/g glucan) and βX
(2mg/g glucan) (CX cocktail) to achieve near-theoretical glucan conversions within 48 h. The
microplates were incubated at 50 ºC with shaking at 250 rpm for 48 h. Sampling was conducted
at 1, 4, 12, 24 and 48 h. The supernatant was then separated from the insoluble solids by filtering
through a 0.45 µm low protein binding hydrophilic microplate based filter (Lot # R6PN00144,
Millipore, Ireland) for protein and sugar analysis. All experiments were carried out in triplicates
with mean values and standard deviations reported in the figures.

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8.2.7

Sugar analysis

Glucose and xylose concentration within the hydrolyzate was analyzed by HPLC with details
provided elsewhere [209].
8.2.8

Quantitation of unbound CBH I, CBH II and EG I

8.2.9

Thermostability of CBH I, CBH II and EG I

0.15 mg/mL of purified CBH I, CBH II and EG I were incubated at 50 ºC. Samples from 0, 1, 4,
12, 24 and 48 hours incubation are evaluated for their specific activities on various substrates
(CBH I and CBH II were tested on Avicel with incubation at 50 ºC for 24 hours; EG I was tested
on carboxymethyl cellulose with incubation at 50 ºC for 1 hour). The reducing sugars were
measured using the 3,5-dinitrosalicylic acid (DNS) based assay [71] . Relative activities are
reported based on samples from 0 hour incubation.
8.3 Results and Discussion
8.3.1

Cellulase hydrolytic activity on different cellulosic substrates

The relative enzymatic digestibilities of various cellulose allomorphs and amorphous cellulose
were very different (Figure 8.1). All substrates were hydrolyzed using a defined equi-mass
cellulase cocktail comprising of CBH I, CBH II and EG I (at 45 mg total cellulase loading/g
glucan). The digestibility of cellulose, during the first 4 hours of digestion, was found to rank in
the following order: Amorphous cellulose or AC (90%) > cellulose III (58%) > cellulose II
(54%) > cellulose I (43%). Figure 8.1E depicts the average hydrolysis rate at different time
points. Near-theoretical glucan conversions were achieved for cellulose I, cellulose II and III,
and amorphous cellulose at 48 h, 24 h, and 12 h respectively. Cellulose I saw the most significant
decrease in its hydrolysis rate (within the first 4h of hydrolysis) among all allomorphs. Cellulose

155

II maintained a similar hydrolysis rate that was marginally higher than Cellulose III and AC at 4h.
These results demonstrate how the recalcitrance of cellulose is closely related to its allomorph.
8.3.2

Cellulase binding to different cellulosic substrates during course of hydrolysis

The extent of unbound cellulases (CBH I, CBH II and EG I) for the various cellulose allomorphs
and amorphous cellulose during saccharification was found to vary considerably. The level of
unbound (or free) CBH I, CBH II and EG I for various cellulosic substrates was estimated using
the depletion method and are shown in Figure 8.1A-D. The initial bound enzyme concentration
(after 1 hour incubation) was calculated by the depletion method, i.e., subtracting free enzyme in
supernatant from total enzyme loaded (Figure 8.2). AC had the highest binding capacity for CBH
I, CBH II and EG I (more than 85% of added enzymes were bound after 1 hour). Cellulose II’s
binding capacity for CBH I and CBH II was greater than 70%, while 60% of EG I was bound
after 1 hour. A greater percentage of the originally added cellulases were bound to AC and
Cellulose II compared to Cellulose I (33-44% of cellulases are bound and EG I bind 10% more
than CBH). On the contrary for cellulose III, only 17% CBH I, 12% CBH II and 6.6% EG I was
bound to the substrate after 1 h of saccharification. This is striking considering that the digestion
rate for Cellulose III is lower only compared to amorphous cellulose. Comparing the levels of
unbound cellulases for amorphous cellulose after 1 h of saccharification and cellulose
allomorphs after 4 h of saccharification is appropriate since the extent of glucan conversions is
comparable. However, the trends noted after 1 h of hydrolysis were also seen for various
cellulose allomorphs (I, II and III) after 4 h of hydrolysis.
For later stages of hydrolysis (24-48 hours), most of the cellulases are present in their unbound
state (>90% CBH I and EG I are present as free enzymes at 48 h) in the supernatant as the
cellulose is depleted (>95% glucan conversion for all substrates). These results are consistent

156

with previous work that reported industrial-scale cellulase production using pure cellulose (e.g.,
milled cotton, sulfite pulp and Solka Floc) to induce enzymes and protein recovery after
complete substrate solubilization [85]. However, after 12 hours of saccharification the level of
unbound CBH II in the supernatant keeps decreasing for all substrates. It’s likely that CBH II has
lower thermostability compared to the other cellulases that causes it to unfold and alter its
structural properties (e.g., pI, molecular weight, amino acid composition) that influences its
detection using the ion-exchange chromatography method. Our method to quantify cellulases is
based on separation of enzymes from background UV-absorbing lower molecular components
based on their molecular weight by gel filtration chromatography followed by individual
cellulase separation and quantification via ion exchange chromatography [228]. If a certain
fraction of CBH II is excluded from the chromatographic steps due to change in its
molecular/structural properties then the total quantifies CBH II will be lower than theoretical
available unbound protein in the supernatant.

157

A
Free enzyme or sugar yield

100%
80%
60%
CBH II

40%

EG I
CBH I

20%

Glucose yield

0%
0

10

20

30

40

50

Hydrolysis Time (h)
Figure 8.1 Sugar yield and the percentage of enzymes in supernatant for Cellulose I (A),
Cellulose II (B), Cellulose III (C) and amorphous cellulose (D) during 48 hour hydrolysis at
50 ºC and comparison of average hydrolysis rate for different cellulose allomorphs (E).

158

Figure 8.1 (cont’d)

100%
Free enzyme or sugar yield

B

80%
60%
40%
20%
0%
0

10

20

30

40

50

40

50

Hydrolysis Time (h)
100%
Free enzyme or sugar yield

C

80%
60%
40%
20%
0%
0

10

20

30

Hydrolysis Time (h)

159

Figure 8.1 (cont’d)

100%
Free enzyme or sugar yield

D

80%
60%
40%
20%
0%
0

10

20

30

40

50

Hydrolysis Time (h)

E
Average hydrolysis rate (h-1)

60%
Cellulose I
Cellulose II

40%

Cellulose III
Amorphous Cellulose

20%

0%
1

4

10
Hydrolysis time (h)

160

24

48

100

Cellulose I
Cellulose III

Cellulose II
Amorphous

Bound enzyme at 1 h

100%
80%
60%
40%
20%
0%
CBH II

EG I

CBH I

Figure 8.2 The level of bound CBH I, CBH II and EG I for different cellulose allomorphs at
1 hour.
8.3.3

Trichoderma cellulases thermostability

In order to confirm previous hypothesis that CBH II tends to easily lose activity during
hydrolysis at 50ºC, purified cellulases were incubated at 50ºC for different time periods and
assayed to check their specific activity. From Figure 3, we can clearly see that ~60% of CBH II’s
original activity was preserved after incubation at 50ºC for 48 hours; whereas, CBH I and EG I
retain more than 90% of their original activity. In the case of Cellulose III, 70% of unbound
CBH II is found in the supernatant after 48 hours, while only 60% of the enzyme is found for the
other substrates (Figure 1). These results suggest that thermal denaturation of CBH II, as
indicated by the loss in enzymatic activity, is responsible for altering the protein structure and
hence impacting protein quantification using the ion-exchange chromatography method. A
noticeable drop in cellulase activity is seen after 12 hour of incubation at 50ºC which is

161

consistent with the corresponding decrease in predicted concentration of unbound CBH II seen in
Fig 1A-D. In the current set of thermostability experiments no substrates was added, however, it
is reasonable to suspect that cellulase activity preserved would be different in a heterogeneous
environment in the presence of cellulose. Binding to cellulose has been reported to stabilize
cellulase activities and prevent thermally-induced denaturation [231, 232]. However, since it is
difficult to directly measure individual bound enzyme’s activity during hydrolysis, these results
offer evidence of extent of activity loss during incubation.Pretreated corn stover hydrolysis

Relative activity

100%

80%
EG I
CBH I
CBH II

60%

40%
0

1

4

12

24

48

Incubation time (h)
Figure 8.3 Relative activity for CBH I, CBH II and EG I after certain time of incubation at
50 ºC.
8.3.4

Cellulase binding and hydrolytic activity on pretreated lignocellulosic biomass

Commercialized large scale hydrolysis for biofuels or chemicals production has to be considered
by operating with pretreated biomass rather than pure cellulose. After pretreatment, biomass is

162

modified physically and chemically to decrease the recalcitrance which facilitated quick sugar
releasing during saccharification. To avoid plant species different, corn stover is chosen as the
single feedstock. AFEX is a “dry to dry” process which does not remove any liquid stream
during pretreatment hence preserves almost all of the composition [103, 209]. Dilute acid
pretreatment, which remove most of the hemicelluloses from liquid stream and part of lignin.
The removal of most hemicellulose modifies the substrate composition. Both cellulose and lignin
are enriched compare to untreated biomass [103]. Ionic liquid pretreatment, a novel pretreatment
technology, swells the cell wall and remove significant amount of lignin from biomass hence the
glucan and xylan component are enriched. These three pretreatments are representatives for
many other alkaline, acid and extraction based pretreatment such as lime, steam explosion and
organosolv. The composition of pretreated corn stover is shown in Table 8.1. AFEX CS has
almost identical composition with untreated one. Dilute acid remove most of the xylan and
consequently has the highest glucan and lignin content. IL pretreatment removes most of the
lignin and preserved most xylan. All of the experiments are performed at 1% standard glucan
loading so their theoretical glucose yields are same. Theoretical xylose and lignin concentration
therefore are quite different from each other.
CBH I, CBH II and EG I are the “work horse” enzymes for biomass saccharification. For AFEX
CS, optimized cocktail need around 70%-80% of those enzymes [71, 74, 198]. Endo-xylanase, βxylosidase and other auxiliary hemicellulases are necessary for hemicellulose hydrolysis to
achieve high xylose yield [205]. To evaluate how xylan component affect cellulases binding, all
pretreated CS experiments are performed in two set of enzymes cocktail. One is only with CBH I,
CBH II, EG I and βG (cellulases, abbreviation of C Cocktail). The other is C Cocktail with
addition of EX and βX (cellulases with xylanases, abbreviation of CX Cocktail).

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Table 8.1 Composition and theoretical concentration of cellulose allomorphs (cellulose I, II,
III and amorphous cellulose) and pretreated corn stover.
Composition
Substrates
Cellulose allomorphs
Untreated/AFEX CS
IL CS
ACID

Glucan
***

~100%
34.6%
46.9%
60.6%

*

Xylan
0
19.6%
29.8%
3.3%

Theoretical concentration (g/L)
Lignin

**

0
11.0%
2.7%
32.9%

Glucose

Xylose

Lignin

11.11
11.11
11.11
11.11

0.00
5.66
7.22
0.62

0.00
3.18
0.58
5.43

*Based on dry weight
**Insoluble Klason lignin
***Non-cellulose component is neglectable
For AFEX CS, as show in Figure 8.4, with the C Cocktail, the glucose yield can get around 80%
after 48 hours while xylose yield is very low (<10%). This is consistent with our previous
experiment results [71, 205]. Addition of EX and βX can enhance the xylose yield to around 54%
and most of the glucan are hydrolyzed into monomeric glucose. The ratios of enzyme in
supernatant are quite different from that in pure cellulose. At 1 hour, most of cellulases are
bound onto biomass. With the saccharification moving forward, only limited amount of enzymes
return to supernatant. CBH I has the highest recovery with 47% and 60% for C and CX cocktail
respectively. For CBH II, much less amount of enzyme can be desorbed from biomass. CX
cocktail can help to increase 8% more CBH II to final 37% in supernatant. For EG I, even in CX
cocktail, only 28% is detected in the supernatant. The xylanases help remove xylan wrapped
around glucan chain hence increase cellulose digestion. Just EX and βX are sufficient to
solubilized xylan with some of xylose in brach substituted oligomeric sugar form. So for CX
cocktail digested AFEX CS, the most of the remaining insoluble biomass is lignin residual. For
AFEX CS, the EG I has the highest unproductive binding level onto lignin and CBH I has the
lowest. But still, quite amount of CBH I (40%) cannot be recovered. Regarding previous
research focused on mixture optimization of CX cocktail on AFEX CS, EG I has the highest
164

ratio counting 31% of the total enzyme loading [71]. The EG I has the highest unproductive
binding level could explain why the optimum cocktail requires so much EG I.

165

Free enzyme or sugar yield

A

100%

CBH II
CBH I
Xylose yield

80%

EG I
Glucose yield

60%
40%
20%
0%
0

10

20

30

40

50

Hydrolysis Time (h)

Free enzyme or sugar yield

B

100%
80%
60%
40%
20%
0%
0

10

20

30

40

50

Hydrolysis Time (h)
Figure 8.4 Sugar yield and the percentage of enzymes in supernatant for AFEX CS with (B)
and without (A) endoxylanase and β-xylosidase during 48 hour hydrolysis at 50 ºC.

166

For ACID CS (Figure 8.5), C cocktail is sufficient to get almost 90% glucose yield. The CX
cocktail slightly improves glucose yield. However, for both cocktail, most of cellulases bound
unproductively after 48 hours hydrolysis. Only around 30% CBH I can be recovered, which is
around half of AFEX CS. Around 20% of EG I and 10% of CBH II remain in the supernatant.
These results suggest the high lignin content in AICD CS contribute less enzyme recovery.
IL CS only contains less than 3% of lignin and the time course hydrolysis and free enzyme assay
results are shown in Figure 8.6. With the C cocktail, more than 80% of glucose yield is obtained
after 48 hours, while the CX cocktail can release around 90% of glucose. It takes 24 hours to
release most the xylose, which is much slower compared to AFEX CS. Most cellulases bound
onto biomass at 1 hour. With the conversion increasing, enzymes return to supernatant with
different extent. CBH II is the highest recoverable enzymes. 60% can be recovered. Regarding
the CBH II lost activity during hydrolysis for pure cellulose allomorphs. It is likely that most of
CBH II can be recovered after hydrolysis. The recovery of EG I in IL CS is only around 30% but
is still higher than AFEX CS and ACID CS. The interesting result is for CBH I. Less than 50%
can be recovered in IL CS, which is less than AFEX CS although the lignin content of AFEX CS
is much higher (Figure 8.7).

167

A
Free enzyme or sugar yield

100%
80%
CBH II
EG I
CBH I
Glucose yield
Xylose

60%
40%
20%
0%
0

10

20

30

40

50

40

50

Hydrolysis Time (h)

B
Free enzyme or sugar yield

100%
80%
60%
40%
20%
0%
0

10

20

30

Hydrolysis Time (h)
Figure 8.5 Sugar yield and the percentage of enzymes in supernatant for ACID CS with (B)
and without (A) endoxylanase and β-xylosidase during 48 hour hydrolysis at 50 ºC.

168

A
Free enzyme or sugar yield

100%

CBH II
Glucose

80%

EG I

CBH I

Xylose

60%
40%
20%
0%
0

10

20

30

40

50

40

50

Hydrolysis Time (h)

B
Free enzyme or sugar yield

100%
80%
60%
40%
20%
0%
0

10

20

30

Hydrolysis Time (h)
Figure 8.6 Sugar yield and the percentage of enzymes in supernatant for IL CS with (B)
and without (A) endoxylanase and β-xylosidase during 48 hour hydrolysis at 50 ºC.

169

80%

Free enzyme at 48 h

CBH II
EG I

60%

CBH I
40%

20%
IL CS

AFEX CS

ACID CS

0%
0

1

2

3

4

5

6

Lignin concentration (g/L)

Figure 8.7 Percentage of enzyme in supernatant after 48 hours hydrolysis of AFEX CS,
ACID CS and IL CS.
How inhibitory lignin is always bewilder researchers to fully understand the hydrolysis process.
Many efforts have been done to understand the lignin’s role. However, most of those researches
are limited from two major drawbacks: (1) they lack the tools to monitor individual enzyme.
Only the total protein concentration is reported [153, 228]; (2) By extract lignin from biomass,
the structure of lignin might be modified significantly hence their inhibitory effect could also has
been changed and this is why some results show little or no inhibitory effect of extracted lignin
[115, 227]. These experiments reveal meaningful results for enzymatic hydrolysis and cellulases
binding. High unproductive binding level of enzymes onto lignin is confirmed. Generally, high
lignin content correlated to high level of unproductive binding. The binding of cellulases is both
governed by substrate composition and crystal structure. Among cellulose allomorphs, different

170

binding levels are observed due to the variation of surface area, surface hydrophobicity, etc. The
stability of each cellulase is different. CBH II is prior to lose activity at 50 ºC whereas CBH I
and EG I still maintain most of their activities after 48 hours incubation.
All these findings and hypothesis shed a light to the future researches which are focused on
understanding hydrolysis mechanism and engineering high efficient hydrolysis systems. For
pretreatment studies, with this techniques applied, rational design and evaluation of a novel
pretreatment are possible by focusing on minimizing unproductive binding of enzyme onto
biomass. Hence the enzymes could be recovered for the following batch of hydrolysis. From the
protein engineering point of view, with above tools, evaluating the mutant enzymes to make
them have less unproductive binding level (for EG I) as well as increase their thermostability (for
CBH II) could help to develop a superior cocktail.
8.4 Conclusion
In this research, we have performed experiments to track CBH I, CBH II and EG I during
enzymatic hydrolysis. Cellulose allomorphs and pretreated corn stover are chosen as substrates
for continuous 48 hours saccharification. The digestibility from highest to lowest for cellulose
allomorphs is amorphous cellulose > cellulose III > cellulose II > cellulose I. The binding
capacity rank from highest to lowest is amorphous cellulose > cellulose II > cellulose I >
cellulose III. For pretreated biomass, AICD CS has high unproductive binding due to its high
lignin content. EG I is much hard to recover compared to CBH I. Removal of lignin by IL
facilitate enzymes recovery especially for CBH II, but less favorable for CBH I compared to
AFEX CS.

171

CONCLUSION AND SUGGESTIONS FOR FUTURE RESEARCH
The fate of cellulosic ethanol production is largely decided by how large quantity of cheap
fermentable sugar can be obtained. The work in this thesis focused on enzymatic hdyrolsys of
pretreated biomass. The major conclusion is
1) Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a
significant reduction in enzyme loading without sacrificing hydrolysis yield.
2) A diverse set of accessory hemicellulases from bacterial sources was found necessary to
enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover.
3) A high-throughput Fast Protein Liquid Chromatography (HT-FPLC) based method has
been developed to quantify CBH I, CBH II and EG I present in hydrolyzates of untreated,
AFEX, and dilute-acid pretreated corn stover. This method can accurately quantify
individual enzymes present in complex binary and ternary protein mixtures without
interference from plant cell wall derived components.
4) The binding studies on pure cellulose allomorphs reveal the binding level is not alway
positive correlate to substrate digestibility. CBH II is less stable during hydrolysis.
Presence of lignin is responsible for significant unproductive cellulase binding.
All these results in this work shed a light to the future research which is focused on engineering
high efficient hydrolysis systems and understanding lignocellulosic biomass hydrolysis
mechanism. The following suggestions and ideas could be helpful for future work.
1) Screening and evaluating novel enzymes for AFEX pretreated biomass. Esterase, GH 61
enzymes and many other possible cocktail candidates are possible to work
synergistically with current cocktail and hence decreasing total enzyme loading.

172

2) Evaluating enzyme cocktail at different pretreatment severity. Pretreatment is normally
evaluated by using commercial enzymes at fixed conditions. Pretreated biomass at
different pretreatment severity may require different combination of enzymes at different
loading dosage. So far, few studies have been focused on this area which is also
important to evaluate pretreatment and hydrolysis economic analysis as an integrated
system.
3) Studing the binding properties of other enzymes. In this work, CBH I, CBH II and EG
I’s binding properties are studied. Other enzymes, such as xylanases, endo-glucanases
are also binding on biomass and it will be interesting to study their binding behaviors.
GH 61 enzymes have no detectable activities and have no effect on pure cellulose/xylan
but they can decrease total enzyme loading significantly on pretreated biomass. Is that
possible that GH 61 enzymes can reduce unproductive binding level of other work horse
enzymes? Is any protein-protein interaction between those enzymes?
4) Understanding the effect of hydrolysis end products and degradation products. During
hydrolysis, sugars (monomeric and oligomeric format) and degradation compounds
formed during pretreatment are released into the liquid phase. At high solid loading
hydrolysis experiment, these compounds are in much higher concentration hence could
inhibit enzyme or affect their binding properties. Studing how these compounds affect
cocktail, enzyme binding could help to improve hydrolysis at high solid loading.
5) Establishing a comprehensive hydrolysis model. With the tools developed in this work,
purified enzymes and their binding level on substrates can be used to build a model
which is integrated by individual enzyme’s parameters. Rather than many other

173

simplified models, this comprehensive model could reveal more important information
during hydrolysis and help to design a more efficient hydrolysis process.

174

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