_.._ N m- op>«.~ . .-...7 .-~.... ..._. THE isome AND CHARACTERIZATiON.‘ OF THE EXTRACELLULA SE11 'R .PROTEA PENICILLIUM ROQUEEFORTI' BP13 Thesis for. the Degree of Ph. D. MICHIGAN STATE UNIVERSITY H. WAYNE MODLER 1973‘ an .1 f. n. C . C .31.. . .. _. L... .....: . u... 11—... .21.. .. .n: 3 1293' 10559 7714 _ a“ LIBRARY Michigan Stan ' University This is to certify that the thesis entitled The Isolation and Characterization of the Extracellular Protease of Penicillium Roqueforti BP-l3 presented by H. Wayne Modler has been accepted towards fulfillment of the requirements for Ph.D. degree in Food Science and Human Nutrition MMSM I Date February 8, 1973 0-7 639 use: my :5 mm; & sons lgOK BINDERY IIIC. ABSTRACT THE ISOLATION AND CHARACTERIZATION OF THE EXTRACELLULAR PROTEASE OF PENICILLIUM ROQUEFORTI BP-l3 BY H. Wayne Modler The extracellular protease of Penicillium roqueforti BP-13 (EC 3.4.4.99) was prepared by shake culturing the fungus in Czapek-Dox broth containing 0.5% of Proteose-Peptone No. 3. A cell free extract (CFE) was prepared and then concentrated by ultrafiltration and pervaporation. The enzyme was isolated from the concen- trated CFE by fractionation over a series of Sephadex columns. The final enzyme preparation contained small amounts of peptides and/or amino acids which were present as either impurities or were formed as a reSult of autolysis. Attempts to remove these small molecular weight components by ion exchange, dialysis, electrodialysis and precipitation with ammonium sulfate were not successful. The BP-13 protease had a pH optimum of 3.0 and 5.5 for bovine serum albumin and casein, respectively. The enzyme exhibited maximum stability to pH in the range of 3 to 6. The optimum temperature for activity was 45-46 C H. Wayne Modler when using 1% casein at pH 5.75; this optimum was based on a 9 min end point assay. An Ea of 8000 cal/mole for the hydrolysis of casein was calculated from an Arrhenius plot. Above 46 C, the enzyme was subject to irreversible first order thermal inactivation. Serine and sulfhydryl protease inhibitors had no significant affect on enzymatic activity when compared to pepsin. Ethylenediaminetetraacetic acid did not alter the activity of the protease. Carboxyl modification with diazoacetoglycine methylester produced over 90% decrease in activity for both the BP-l3 protease and pepsin, indi- cating the presence of aspartic and/or glutamic acid at the active site of the BP-13 protease. Of 15 peptides and amino acid esters, only L- leucyl-L-tyrosine was hydrolyzed to any detectable degree. Even with this substrate, hydrolysis was too slow to be of value in an assay procedure. The action of the enzyme on the oxidized B chain of insulin resulted in the formation of 14 ninhydrin areas on a peptide map. This was in- terpreted to be an accurate reflection of the number of peptide bonds hydrolyzed when the very proteolytic nature of the enzyme is taken into consideration. The two major casein components, as- and B-casein, were extensively hydrolyzed and no longer identifiable on polyacrylamide gel patterns of whole casein after 20 hr (30 C, pH 5.75) incubation with the BP-13 protease. Kappa casein appeared to remain relatively unchanged. When the H. Wayne Modler release of trichloroacetic acid (TCA) soluble nitrogen from a casein solution was followed over a period of 1 hr, striking differences were evident: the calf rennet dis- played an initial specificity for the methionyl-phenylalanine linkage of K-casein, followed by very slow and non-specific hydrolysis of casein in general. The BP-13 protease produced a rapid and nearly linear increase in TCA soluble nitrogen over the same time period. Milk clotting studies revealed that the fungal protease was 14 times as pro- teolytic as calf rennet for the same milk clotting activity. Milk never formed a smooth firm gel but tended to curdle. This was followed by syneresis and precipitation of the casein from suspension. THE ISOLATION AND CHARACTERIZATION OF THE EXTRACELLULAR PROTEASE OF PENICILLIUM ROQUEFORTI BP-13 BY A H9WWayne Modler A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1973 ACKNOWLEDGMENT S The author wishes to express sincere appreciation to Dr. C. M. Stine and Dr. J. R. Brunner for their council and guidance throughout the course of this graduate program. Appreciation and thanks are extended to members of the guidance committee, Drs. H. A. Lillevik, Department of Biochemistry and E. S. Beneke, Department of Botany and Plant Pathology for their advice and effort in reading this manuscript. The author is indebted to Dr. R. F. McFeeters for his many helpful suggestions with regard to experimental procedures and interpretation of results. The technical assistance of Miss Ursula Koch is also gratefully appreci- ated. Special thanks are extended to Drs. T. Niki and Y. Yoshioka of Snow Brand Milk Products, Japan, for supplying the Penicillium rogueforti culture used through- out this study. Financial support provided by the Department of Food Science and Dairy Research Incorporated is gratefully acknowledged. ii The author is especially grateful to his wife, Linda Jane, for her understanding and encouragement. He also wishes to acknowledge the inspiration provided by his parents. iii TABLE OF CONTENTS Page INTRODUCT ION O O O O O O O O O O O O O O 1 LITERATURE REVIEW . . . . . . . . . . . . 3 Ripening of Blue Type Cheeses. . . . . . . . 3 Variation of Proteolysis in Relation to Strain . . 4 Media for Culturing g. rogueforti . . . . . . 6 Characteristics of P. ro ueforti Protease. . . . 7 Nomenclature of Rennet and Rennet Substitutes . . 9 Criteria for Choosing Calf Rennet Substitutes . . 10 Calf Rennet Substitutes. . . . . . . . . . 13 Animal O O O O O I O O O O O O O O O 13 Calf Rennet-Pepsin Blends. . . . . . . . 13 Bovine Pepsin. . . . . . . . . . . . 14 Fungal Rennets . . . . . . . . . . . . 15 a. USillus O O O O O O O O O O O O 15 E. para31t1ca. . . . . . . . . . . . 17 a. miehei O O O O O O O I O O O O O 18 Comparison of Fungal Proteases . . . . . . . 19 Bitterness Associated With Calf Rennet Substitutes . . . . . . . . . . . . . 23 Potential Calf Rennet ubstitutes . . . . . . 25 Bacterial Rennets . . . . . . . . . . . 25 Bacillus Species. . . . . . . . . . . 25 Fungal Rennets . . . . . . . . . . . . 27 Aspergillus niger . . . . . . . . . . 27 Basidomycetes. . . . . . . . . . . . 27 Plant Rennets . . . . . . . . . . . . 27 iv Papain O O O O O O O O I O I FiCin I O I O O I O O O O O cardoon. O O O O O O O O O I Microbial Rennets in General . . . . . Additional Calf-Rennet Substitutes . . . EXPERIMENTAL PROCEDURES . . . . . . . Microbiological Techniques. . . . . . Selection of Cultures. . . . . Detection of Proteolytic Activity. . Type I . . Type II. . Type III . Type IV. . Standard Plate Counts. . . . . . . Spore Counting . . . . . . . . . Shake-Culturing. . . . . . . Preparation of Cell Free Extract (CFE) . Preparative Procedures . . . . . . . Buffers O O O O O O O O O O 0 Universal Buffer. . . . . . . . Additional Buffers . . . . . . . casein. . O O O O O O O O O 0 Sodium Caseinate. . . . . . . . Hammersten Casein . . . . . . . Enzyme Substrate. . . . . . . Dialysis Tubing. . Dialysis . . . . Pervaporation . . Lyophilization . . Standardized Assay Procedure for Proteolytic ACtj-Vity O O O O O I O O O I O Assay Procedure. . . . . . . . . Casein Standard Curves . . . . . . Page 27 28 28 29 3O 31 31 31 31 32 32 32 33 33 33 34 34 34 34 35 35 35 36 36 36 37 37 37 38 38 Page Bovine Serum Albumin (BSA) Standard Curves . . 39 Enzyme Unit . . . . . . . . . . . . 39 Determination of Variables Involved in Assay . . 40 Optimum pH. . . . . . . . . . . . . 40 Stability to pH 0 O O O O C O O O O O 40 Optimum Temperature. . . . . . . . . . 40 Stability to Temperature . . . . . . . . 40 Effect of Substrate Concentration . . . . . 41 Study of Autolysis . . . . . . . . . . 41 Purification of BP-13 Protease. . . . . . . 41 Precipitation of Enzyme . . . . . . . . 41 SOlventS. O O O O O O O O O O O O 41 salt 0 O O O O O O O O O O O O O 42 Ultrafiltration . . . . . . . . . . . 42 Gel Filtration . . . . . . . . . . . 42 Preparation of Sephadex. . . . . . . . 42 Application of Sample . . . . . . . . 43 Chromatography. . . . . . . . . . . 43 Removal of the Nucleic Acids. . . . . . . 44 Further Purification of BP-13 Protease . . . . 45 Ion Exchange . . . . . . . . . . . . 45 Preparation of Celluloses . . . . . . . 45 Preparation of Resins . . . . . . . . 45 Preliminary Ion Exchange Experiments . . . 46 Ion Exchange Experiments . . . . . . . 46 Electrodialysis . . . . . . . . . . . 47 Analytical Procedures. . . . . . . . . . 47 Protein. . . . . . . . . . . . . . 47 Lowry-FOIID. o o o o o o o o o o o 47 Kjeldahl. O O O 0 O O O O O O O O 48 vi Carbohydrate . . . . Acrylamide Gel Electrophoresis . Alkaline Gels . . . . Acid Gels . . . . Sodium Dodecyl Sulfate (SDS) Gel .Electro- phoresis for Determination of Molecular weight. a o o o o Staining and Destaining of Acrylamide Gels Amido Black 108 . . . Coomassie Blue. . . . Absorption Spectra . . Analysis of Insulin (B Chain) Hydrolysates Hydrolysis . . . . High Voltage Electrophoresis Descending Paper Chromatography Hydrolysis of Synthetic Substrates. Peptides. . . . . . Esters O O O C O 0 Determination of Molecular Weight Filtration . . . . . Enzyme Inhibitors . . . . Serine Protease Inhibitor. Sulfhydryl Inhibitors . . Carboxyl Inhibitor . . . Synthesis . . . . . Reaction With Enzymes . Effect of EDTA and Calcium Milk Clotting and Proteolytic Activity Protease . . . . . . Milk Clotting. . . . . TCA Soluble Nitrogen . . Gel Electrophoresis of Casein Hydrolysates Evaluation of Milk Clotting Ability vii of Page 48 48 49 50 51 51 51 51 52 52 52 52 53 53 54 54 55 55 55 56 57 57 57 58 58 58 58 59 59 Page RESULTS AND DISCUSSION . . . . . . . . . . 61 Enzyme Production . . . . . . . . . . . 61 Detection of Proteolysis . . . . . . . . 61 Choice of Broth for Shake Culturing . . . . 63 Shake-Culturing Experiments . . . . . . . 63 Determination of Assay Parameters. . . . . . 70 Effect of Substrate Concentration and pH on Protease Activity. . . . . . . . . . 70 Optimum pH and Stability to pH . . . . . . 72 Optimum Temperature and Stability to : Temperature. . . . . . . . . . . . 75 Activation Energy for Hydrolysis of Casein . . 82 Purification of BP-13 Protease. . . . . . . 83 Concentration. . . . . . . . 83 Purification of the BP- 13 Protease by Gel Filtration . . . . . . . . . . . . 87 Absorbance Spectrum of Nucleic Acid-Protein and Enzyme Peaks. . . . . . . . . . . 96 Standard Plate Counts. . . . . . . . . . 96 Electrophoresis of Various Fractions Resolved by Gel Filtration . . . . . . . . . . 98 Further Purification of the BP-13 Protease . . 103 Determination of Molecular Weight. . . . . . 111 Inhibition Studies. . . . . . . . . . . 115 Serine Protease Inhibitor. . . . . . . . 115 Sulfhydryl Inhibitors . . . . . . . . . 117 Carboxyl Modification . . . . . . . . . 119 Effect of EDTA and Calcium . . . . . . . 120 Hydrolysis of Synthetic Peptides and Amino Acids Esters by BP-13 Protease . . . . 120 Hydrolysis of the Oxidized B Chain of Insulin O O O O O I O I I O O O O 123 Comparison of the Proteolytic Action of the BP-13 Protease and Calf Rennet . . . . . . 126 Evaluation of Milk Clotting Ability . . . . 130 viii (Page SUMMARY AND CONCLUSIONS . . . . . . . . . . 132 BIBLIOGRAPHY. . . . . . . . . . . . . . 133 APPENDIX 0 O O O O O C O O C C O O O O 150 ix Table 1. 10. 11. LIST OF TABLES Qualitative Detection of Proteolysis by P. ro ueforti (BP-13) When Grown on Various Types 0 Me 1a . . . . . . . . . . Effect of Media and pH on the Growth of 3. quueforti (BF-13) o o o o o o o o 0 Recovery of BP-13 Protease Activity After Precipitation With Various Reagents . . . Enzyme Purification Summary of BP-l3 Protease O O O O O O I O O O O 0 Standard Plate Count of BP-13 Protease During Purification Procedure . . . . . . . Effect of Repeated Chromatography on Specific Activity of BP-13 Protease . . . Effect of Electrodialysis on Specific Activity of BP-13 Protease . . . . . . Characteristics and Method of Regeneration of Ion Exchangers Used in the Purification Of BP-13 PrOtease o o o o o o o o 0 Residual Activity in Supernatant After Mixing of BP-13 Protease With DEAE and SE Cellulose at Various pH Levels. . . . . Recovery of BP-13 Protease Activity and Protein From Cellulose Ion Exchangers When Eluting With Citrate Buffer at Various pH Levels and Ionic Strengths . . . . . . Hydrolysis of Synthetic Peptides and Amino Acid Esters by BP—13 Protease . . . . . Page 62 64 85 94 98 104 108 110 111 112 122 Table Page 12. Comparison of Milk Clotting Activity and Proteolytic Activity of Calf Rennet and BP-‘13 Pretease o o o o o o o o o o 131 A-l. Protocol for Preparation of 0.04 M Universal Buffer . . . . . . . . . 153 A-2. Summary of BP-13 Protease Properties . . . 154 xi LIST OF FIGURES Figure Page 1. Effect of Tryptone on Production of Extra- cellular Protease by P. rogueforti (BP-13) When Shake CuItured 1n Czapek- Dox Broth . . . . . . . . . . . . 65 2. Effect of Proteose-Peptone No. 3 on Production of Extracellular Protease by P. rogueforti (HP—13) When Shake Cultured In Czapek-Dox Broth. . . . . . . . . 66 3. Production of Extracellular Protease by Various Strains of E. rogueforti During Shake Culturing in Czape -Dox Broth Containing 0.5% Proteose—Peptone No. 3 . . 67 4. Production of Extracellular Protease and Change in pH by P. ro ueforti (BP-13) During Shake Cultur1ng 1n Czapek-Dox Broth Containing 0.5% Proteose-Peptone No. 3 . . 69 5. Increase in TCA Soluble Nitrogen (Lowry- Folin) Resulting From Hydrolysis of Casein by the BP-13 Protease (CFE) at 30 C . . . 71 6. Activity of BP-l3 Protease (CFE) at Various ph Levels Using 1% BSA as Substrate . . . 73 7. Activity of BP-13 Protease (CFE) at Various pH Levels Using 1% Casein as Substrate . . 74 8. Stability of BP-l3 Protease (CFE) to pH. . . 76 9. Relative Activity of the BP-13 Protease (CFE) at Various Temperatures . . . . . . . 77 10. Rate of Hydrolysis Of Casein at Various Temperatures by the BP-l3 Protease (CFE). . 78 xii Figure 11. Stability of Purified BP—13 Protease to Temperature (Enzyme Incubated 30 min for Temperatures up to 45 C; Above 45 C, Samples Were Incubated 9 min) . . . . . . . . 12. Rate of Thermal Inactivation of BP-l3 Protease at Various Temperatures . . . . . . . l3. Arrhenius Plot for Purified BP-13 Protease Using 1% Casein (pH 5.75) a Substrate. . . 14. Gel Filtration Equipment Used to Purify the BP-l3 Protease: Monitor, Fraction Collector, Recorder, Columns (Two 2.5 x 45 cm, One 1.6 x 100 cm) and Ancillary Equipment. . . 15. Schematic of Gel Filtration Equipment Used for Purifying BP-l3 Protease. . . . . . 16. Purification of BP-13 Protease by Gel Filtration With 650 and G100 Sephadex. . . l7. Purification of BP-l3 Protease by Gel Filtration With G100 Sephadex . . . . . 18. Steps in the Purification of BP-13 Protease . l9. Absorption Spectrum of Nucleic Acid--Protein (GSO/GlOO Sephadex--Peak l) and Purified BP-13 Protease (GlOO Sephadex-—Peak l) . . 20. Alkaline Acrylamide Gel Electrophoresis of Fractions Collected From GSO/GlOO Sephadex . 21. Alkaline (ALK), Acid and 805 Acrylamide Gel Electrophoresis of Purified Enzyme Preparation . . . . . . . . . . . 22. The Affect of 2-Mercaptoethanol (M) on the BP-13 Protease (E) o o o o o o o o o 23. Change in Characteristics of Purified BP-13 Protease After Incubation in 0.04 M Citrate Buffer (pH 5.0, u = 0.3) for 30 hr at 30 C . 24. Descending Paper Chromatogram of Purified Protease After Various Treatments . . . . xiii Page 80 81 84 89 90 91 93 95 97 99 101 102 105 106 Figure Page 25. Electrodialysis Equipment Used in Removing Peptides and Amino Acids From BP-13 Protease . . . . . . . . . . . . 109 26. Standard Curve for Determining Molecular Weight by Gel Filtration Using G-100 Sephadex (1.6 x 100 cm column). . . . . 114 27. Affect of Phenylmethyl Sulfonylfluoride on the Activity of Pepsin, BP-13 Protease and TrypSin O O O O O O O O O O O O 116 28. Affect of Iodoacetamide and P- Chloromercuribenzoate on the Activity of Pepsin, BP-13 Protease and Papain. . . . 118 29. Affect of Diazoacetoglycine Methylester on the Activity of Pepsin, BP-13 Protease and Trypsin . . . . . . . . . . . 121 30. Separation of Insulin (Oxidized B Chain) Hydrolysates Using High Voltage Electro- phoresis Followed by Descending Chroma- tography . . . . . . . . . . . . 124 31. Hydrolysis of 1% Casein (pH 5.75) With Calf Rennet and BP-13 Protease . . . . . . 127 32. Alkaline Acrylamide Gel Electrophoresis of Casein (C) and BP-13 Hydrolysates of Casein at Various Time Intervals (M--min; H--hr). . . . . . . . . . . . . 129 xiv INTRODUCTION The ripening of "Blue Type" cheeses relies mainly on the lipolytic enzyme system of Penicillium roqueforti according to most literature reports to-date. The typical peppery or piquant flavor of a good quality "Blue Type" cheese is due mainly to the oxidation of the fatty acids, by the spores of P. roqueforti, to methyl ketones, alcohols and minor flavor compounds. This area has been investigated in some depth and is well documented in the literature. The proteolytic enzyme system of P. roqueforti was known to exist as early in the twentieth century. However, the importance of this system in the ripening of "Blue Type" cheeses has received only a cursory examination. The main purpose of this investigation was to isolate the extracellular protease or proteases from a. roqueforti and characterize the enzyme(s) with respect to substrate specificity, proteolytic nature (serine, sulfhydryl or acid protease etc.) and its affect on the major casein components of milk (as-, B-, and K-casein). Parameters such as molecular weight, optimum pH, stability to pH, optimum temperature, stability to temperature and the affect of substrate concentration were an essential part of this investigation. In addition, the milk clotting ability and proteolytic activity of this enzyme were to be evaluated and compared to calf rennet. LITERATURE REVIEW The term "Blue" as applied to cheese refers to the blue or blue green veining which permeates the curd of certain mold-ripened cheese. Cheeses which are classified as "Blue" are ripened by the mold Penicillium roqueforti. Such cheese may take on one of many names depending on the country of origin: Stilton, United Kingdom; Gorgonzola, Italy; Roquefort, France; Cabrales, Spain; and Bleu or Blue, Denmark. These are only a few of the Blue type cheeses listed by Scott (1968). In North America, the term Blue is used to describe such cheese manufactured within this hemisphere. Ripening of Blue Type Cheeses Thibodeau and Macy (1942) indicate the typical flavor in Blue cheese is not solely a matter of hydrolysis Of fat; this is only the first step of a much more complex ripening process. Subsequent oxidation of fatty acids to methyl ketones results in the piquant flavor characteristic of Blue cheese. When properly coordinated with the partial hydrolysis of protein by the proteolytic enzymes of g. roqueforti, a good quality Blue cheese is obtained. Attempts to hasten the ripening of Blue cheese by adding lipase should also be balanced by adding protease at the same time according to Thibodeau and Macy (1942). These authors also suggest incorporating the lipase and protease enzymes of P. roqueforti into the curd of Blue cheese, providing these enzymes could be obtained in sufficient quantity to be of benefit. The addition of 6.0 g of mycelium per five 1b of cheese was found to shorten the ripening period by 50%. Thibodeau and Macy (1942) noted that lipolytic and proteolytic enzymes are not readily liberated from the mycelium and suggested that such enzymes should be extracted, isolated and added to the curd in a more readily available form. This presumably would effect an even shorter curing time. Variation of Prgteolysis in Relation to Strain Funder (1949) found a wide variation in the proteolytic properties of various strains of P. roqueforti. The difference in enzymatic properties was so great that extremely different results were obtained in cheese making experiments. Often the difference between morphologically similar strains was greater than between two widely differ- ent species of Penicillium. This author suggests that more attention should be paid to the physiological variation of the molds, their formation of mycelium and the reaction products formed during ripening of the cheese. Proks 23 31. (1956) also noted a large difference in the proteolytic activity of nine strains of P, £23237 Eggti isolated from good Roquefort cheese. The strains differed not only in quantity but also in the types of amino acid released when incubated with casein. Salvadori st 31. (1962a) divided 19 strains of l P. roqueforti into three groups according to amino acid patterns obtained by two dimensional chromatography of casein hydrolysates. Based on proteolytic activities, this author established criteria for choosing a mold intended for use in the dairy industry. In a related series of experiments, Salvadori (1962b) studied three strains of P. roqueforti displaying weak, intermediate and strong proteolytic activity. Using paper chromatography and other techniques, Salvadori showed that histidine and methionine increased the proteolytic activity of all three strains. Great importance was attached to selecting the proper strain of Penicillium for the manufacture of high quality Gorganzola cheese. Niki et_31. (1966) observed that strains of E. roqueforti possessing low lipolytic activity, exhibited high proteolytic activity and vice versa. The most suitable strain for manufacturing blue cheese was found to be one which had moderate proteolytic and strong lipolytic activity. Media for Culturing P. roqueforti As early as 1910, Dox used a liquid medium commonly known as Czapeks broth to surface culture 3. rggueforti. This medium consisted of: 1000 g water, 0.5 9 magnesium sulfate, 1.0 g dipotassium phosphate, 0.01 g ferrous sulfate, 2.0 g sodium nitrate and 30 9 sucrose. The mold was found to grow satisfactorily on this medium and was harvested after 10 days. Naylor 35 31. (1930) found the medium best suited for growth of g. roqueforti and production of protease, contained in 1000 m1 of solution: 0.5 g magnesium sulfate, 1.0 g dipotassium phosphate, 0.5 g potassium chloride, 0.01 g ferrous sulfate, 1.61 g ammonium chloride, 2.5 9 sucrose and 10 g casein. The pH was adjusted to 5.6 and the mold allowed to grow on the surface of the medium for 10 days at 30 c, The mold mat was removed and the filtrate checked for activity. Thibodeau and Macy (1942) noted that while some molds thrive on Czapek broth, 3. rgqueforti grows very poorly when surface cultured. Sodium nitrate, the nitrogen source in Czapeks solution, was one of the poorest sources of nitrogen for g. roqueforti. A modified Czapeks medium, containing less sodium nitrate and added skim milk, was found to satisfy more adequately the requirements for abundant growth and the formation of a thick resistant felt. The maximum proteolytic activity was obtained as soon as the culture had reached the stage of full sporulation. Meyers and Knight (1958) developed a synthetic medium for the submerged growth of a. rgqueforti. This medium contains the same basic ingredients as Czapek-Dox broth, except that sodium nitrate was replaced with ammonium sulfate, Oleic acid was added and a small amount of trace metals included. For optimum growth the initial pH of the medium was adjusted to 4.0. Niki 2E|al. (1966) grew 3. roqueforti on rennet whey at 20 C for seven days. For protease preparation, the whey was acidified with lactic acid to a pH of 4.0 Characteristics of P. roqueforti Protease Naylor (1930) surface cultured P. rgqueforti and checked the protease activity of the filtrate by incubating with casein for 48 hr at 30 C. Activity was based on the recovery of casein precipitated from solution as determined by the Kjeldahl method. The optimum pH for protease activity was 5.3 at 30 C. Thibodeau and Macy (1942) found the optimum pH to be in the range of 5.8 to 6.3 for the protease of g. roqueforti. At this time the enzyme was thought to be trypsin-like but there was some controversy as to the true nature of the protease. The author states that the enzyme is not of the pepsin type and only one protease exists in the mycelium. When this fungus was surfaced cultured, Thibodeau and Macy (1942) noted the protease was released into the culture medium only when autolysis was occurring in the mycelium. These authors postulated that lipases and proteases are difficult to obtain in solution, possibly because they are absorbed on the walls of the cells and are liberated only when the mycelial tissue is disrupted. The protease was precipitated by half saturation with ammonium sulfate. Nishikawa (1957) reported the pH optimum for proteolytic activity of P. roqueforti protease to be 5.5 to 6.0 when cultured at 40 C and assayed in the presence of 0.6% casein sol. The enzyme was stable in the pH range of 5.0 to 6.0, but lower on either side of these limits. Maximum activity was obserVed at 40 C and at 45 C the enzyme was partially inactivated. Niki 25 El: (1966) found that P. roqueforti produced an extracellular protease when grown on rennet whey at pH 4.0. In addition, an intracellular protease was also recovered from the mycelium when disrupted. Both proteases were found in varying quantities in different strains of P. roqueforti. One strain, designated BP-l3, had the highest amount of proteolytic activity. Both the intracellular and extracellular proteases of the BP-l3 strain had a pH optimum of 5.5 with the latter protease having a much narrower range of activity than the intra- cellular protease. Niki £5 31. (1966) states the extra- cellular protease contributes to casein breakdown for a short period during the initial stages of ripening of Blue cheese, while the intracellular enzyme is active during the entire ripening period. Motoc (1970) studied the proteolysis of a 1% solution of Hammersten casein by a preparation of pro- teolytic enzymes obtained by isopropanol extraction of a dried culture of P. roqueforti. Proteolysis was found to be greatest at pH 5.5 and 25 C. Some of the free amino acids released from casein by this enzyme included arginine, serine, aspartic acid, valine and norvaline. Nomenclature of Rennet and Rennet Substitutes The term rennet, as used today, refers to the enzyme system extracted from the fourth stomach or abomasum of a suckling calf. Rennin is the major enzyme in this system but as the calf ages, the ratio of rennin to pepsin decreases. Foltmann (1970) refers to the major milk-clotting enzyme from the abomasum of young calves as chymosin rather than rennin. He indicates that there are three reasons for using the term chymosin: the word rennin resembles renin and leads to confusion with the latter enzyme; more papers are published about renin; and, the name chymosin is 50 years older than rennin. Sardinas (1968) defined rennet as any crude enzyme preparation of animal, plant or microbial origin which curdles milk. Pure milk clotting enzyme ESE gs, regardless of origin, were designated as rennin. The nomenclature of 10 Sardinas will be used in this dissertation with the exception that chymosin will refer specifically to calf or veal rennin. Unless otherwise designated, pepsin will refer to the extract from porcine stomach. Criteria for Choosing Calf Rennet Substitutes Sardinas (1968), Kikuchi and Toyoda (1970) noted that the supply of calf rennet varied seasonably and is becoming progressively more scant as the result of in- creased cheese production, decreasing slaughter of calves and diminished exports of calf rennet to the USA by countries which retain the enzyme for home use. The non-acceptability of products of animal origin to vegetarian populations, particularly in countries like India and Israel, has also stimulated research for microbial and plant rennet substitutes according to Babbar gt_al. (1965) and Dewane (1960). This latter writer states that a calf rennet substitute should have the following charac- teristics: a. Yield of curd comparable to rennet b. Curd should possess physical properties comparable to rennet curd c. Loss of fat in the whey should be minimal d. No detectable flavor defects Vanderpoorten and Weckx (1972) state that the microbial rennets, whose proteolytic prOperties most closely resemble those of veal rennet, offer the best chances of 11 success in cheese manufacture. The microbial protease should act in a similar manner during all three phases of action on casein: a. Phase one is very specific and is designated the primary reaction. Calf rennet cleaves a glyco- macropeptide from K-casein resulting in destabi- lization of the "calcium sensitive aS-casein." b. During the second phase the casein precipitates. c. The third phase is slow and consists of unspecific proteolysis. This phase progresses simultaneously with the primary and secondary reaction and during the ripening of the cheese. Schulz and Thomasow (1970) consider coagulation of milk to be only a small part of the role of the coagulating enzyme in cheese making. These workers attach greater importance to the breakdown of a- and B-casein and other proteins in cheese ripening. In the estimation of these authors, two properties need to be considered when evalu- ating calf rennet substitutes; the breakdown of K-casein in the milk and hydrolysis of proteins in the cheese. The breakdown of K-casein is evaluated by determining the rennet strength. Additional criteria of coagulating properties are elasticity of the coagulum and the ability to separate the curd from the whey. Hydrolysis of the a- and B-casein depends on the nature of the coagulum and the type of cheese being manufactured. 12 Schulz and Thomasow (1970) also stipulate that the nature of the cheese being manufactured dictates the requirements of calf rennet substitute. For example, in butter or cream cheeses which are ripened at 7 C, the coagulum plays an essential role in maturation. With a cheese such as Emmental the curd is scalded in the whey to temperatures of 50 to 58 C for 20 to 40 min which results in partial destruction of the calf rennet added. In this case, thermolabile rennet substitutes will have little influence on protein breakdown with maturation depending mainly on the enzymes of thermobacteria and propionic acid organisms. Kylé-Siurola and Antila (1970) confirmed these results when the fungal rennets of Mucor pusillus Lindt and Endothia parasitica were used. Schulz and Thomasow (1970) also noted that in soft cheeses such as Camembert, Tilsit and Limburger, smaller amounts of rennet are generally used with ripening being primarily a function of the organism smeared on the surface of the cheese. The bitterness often associated with calf rennet substitutes may be produced as a result of a different kind of casein breakdown or increased hydrolysis of whey protein (Schulz and Thomasow, 1970). Temperature of ripening, pH and minerals may also affect the production of bitter flavors. On the other hand, the increased proteolytic effect of rennet substitutes is important for accelerated cheese ripening. In View of this, Schulz and Thomasow (1970) feel that future research should be concerned more 13 with proteolysis by calf rennet substitutes since this may in turn lead to quicker ripening as well as improved cheese flavor. In the future, veal rennet substitutes may have to be designed for a particular variety of cheese, rather than one rennet for all cheese types. Calf Rennet Substitutes Animal Calf rennetepepsin blends.-—Babel (1967); Chapman and Burnett (1968); Thomasow (1971a); Dan and Jespersen (1970); and Emmons £3 21’ (1971) reported similar results when using pepsin-calf rennet blend. Cheese prepared with such a mixture (1:1) of these milk coagulants graded com- parable to cheese manufactured with calf rennet. Pepsin alone was not considered an ideal milk coagulant and ripening agent according to Mickelsen and Fish (1970). Ernstrom (1961) demonstrated the sensitivity of porcine pepsin to pH change in milk while other workers determined proteolysis by chymosin (El-Negoumy, 1968), calf rennet (Sherwood, 1935), and pepsin (Melachouris and Tuckey, 1964; and Sherwood, 1935) in milk, and/or cheese. Results showed pepsin to be less proteolytic than calf rennet or chymosin. Dan and Jespersen (1970) reported the chief objection to the use of procine pepsin, when used as the sole coagulant, have been defects of taste, consistency 14 and texture of the resultant cheese. He also noted a slower breakdown of the casein in cheese produced with a 1:1 mixture of calf rennet and porcine pepsin than in a cheese made with only calf rennet. Both El-Negoumy (1968) and Ledford st 31. (1968) Observed that B-casein is evidently more resistant to calf rennet proteolysis than aS-casein. Cerbulis EE.E£- (1960) noted that pepsin does not hydrolyze B-casein extensively. Itoh and Thomasow (1971) reported that pepsin had the lowest proteolytic activity on casein fractions when com- pared to calf rennet and other microbial rennets. Procine pepsin hydrolyzes most of the protein substrates strongly at its pH optimum of about 2.0 but Fruton (1970) demon- strated that pepsin did not react strongly as a protease above a pH of 6.0. However, pepsin reacts easily on K-casein near pH 6.5 and converts it into "para K-casein" which subsequently causes clotting in milk according to Fruton (1970). Melachouris and Tuckey (1964), and Veringa (1961) believe that these properties are the reason for the somewhat slow ripening of the pepsin cheeses. Sherwood (1935) found that it took three times as much porcine pepsin as calf rennet to produce the same total protein breakdown. Bovine pepsin.--Fox and Walley (1971) made cheese using commercial calf rennet, commercial mixture of calf rennet/pepsin (1:1) and bovine pepsin. All cheese graded 15 special and no signs of bitterness were evident in the cheeses. Bovine pepsin produced the slowest formation of soluble nitrogen. Electrophoretic patterns of Cheddar cheese were identical after 11 months of ripening, sug- gesting similar proteolysis had occurred in each cheese. The bovine pepsin did show one additional well resolved peptide not present with either of the other two coagulating agents. Bovine pepsin is currently being used in combi- nation with calf rennet as a milk coagulant for cheese manufacturing in Canada (Emmons, 1972). Fungal Rennets The molds Mucor pusillus Lindt, Mucor miehei and Endothia parasitica are currently being used to produce microbial rennets on a commercial basis as a substitute for calf rennet. M. pusillus.--Tuasaki gt al. (1967a, 1967b) studied the properties of the crude chymosin-like enzyme from M. pusillus F-27. This acid protease had a pH optimum of 3.5 for digesting casein. The milk clotting and proteolytic activity of this enzyme resembled that of calf rennet more so than most proteases of fungal origin. In addition, the enzyme was more heat stable and more resistant to pH changes than its traditionally used counterpart. Milk clotting activity was affected by calcium (Ca++) ion concentration to a greater extent than was calf rennet. Curves showing the release of trichloroacetic (TCA) soluble 16 nitrogen indicated the fungal rennet was less specific than calf rennet. Moving boundary electrophoretic patterns of aS-casein were similar. Richardson 23 21. (1967) found the rennet extract from M. pusillus produced a greater increase in non-protein nitrogen (NPN) in casein sols and in cheese, than either pepsin or calf rennet. Further work revealed the fungal protease activity was dependent on (Ca++) concentration to a greater extent than calf rennet. Normal curd tension could be restored by adding 0.015% calcium chloride to the cheese milk or by adding 10 to 12% additional microbial rennet. The difference in curd tension could also be obviated by using a longer set time. Curd tension can also be increased by raising the "setting" temperature; however, as the temperatures are increased the fungal rennet clot tends to become progressively weaker after a critial maximum Of 32.2 C is reached. When Cheddar, Brick and Parmesan cheese were manufactured by Richardson gt 31. (1967) employing the fungal rennet of M. pusillus, an increase in the fat content of whey from 0.30 to 0.42% was observed. This is apparently related to slow set and fragility of the curd. Richardson gt 21' (1967) found the protease extract of M. pusillus to be very stable in the dry form or in saline solution; however, severe loss in activity was observed when this enzyme was blended and stored with calf l7 rennet in the liquid form. Results indicate the fungal rennet hydrolyzed the veal rennet. Trop and Pinsky (1971) found that when chymosin and M. pusillus rennin were mixed together, the coagulation activity increased over 200%. This author concludes that one enzyme system appears to be a synergist to the other, suggesting two separate coagulating mechanisms. Itoh and Thomasow (1971) reported that the protease of M. pusillus caused no significant increase in NPN when incubated with as- and B-casein but demonstrated by starch gel electrophoresis that this enzyme degraded as- and 8- casein more than did chymosin or pepsin. E. parasitica.--Sardinas (1968) compared fungal rennet from E. parasitica to animal rennet and found striking similarities with respect to molecular weight, amino acid composition, isoelectric point, pH stability and clotting activity. In addition, this fungal protease was mildly proteolytic, which is an essential attribute for the production of high quality aged cheese. Berridge (1954) noted that when animal rennet is added to bovine milk below 15 C, no curd is formed, though some alteration of the milk occurs. If the temperature of this milk is increased to 37 C, the milk will quickly clot. The protease of M. parasitica acts in an identical manner, according to Sardinas (1968). 18 Shovers and Bavisotto (1967) manufactured a wide variety of cheeses with partially purified M. parasitica rennin and found all products to be equal or superior to control cheeses made with animal rennet. Alais and Novak (1970) confirmed these results, although some differences in coagulating and proteolytic activity were observed. Morris and McKenzie (1970) manufactured Cheddar cheese using microbial rennet derived from M. parasitica. Coagulation times were slightly prolonged when greater than 50% substitution of calf rennet was made. In addition, cheese manufactured with 75% microbial rennet and 25% calf rennet graded slightly lower than controls manufactured strictly from calf rennet. Nadassky (1972) manufactured Edam cheese from "sure curd" (E. parasitica rennet) and found the course and duration of renneting were the same for both types of coagulants. On this basis, the rennet from M. parasitica was considered suitable for use in Dutch type cheese manufacture. M. miehei.--A recent report by Thompson (1972) indicates that M. miehei rennet has commercial significance. This author manufactured Cheddar cheese from Mucor rennet and found this enzyme preparation to be equal to calf rennet in all respects: clotting and cutting time, curd firmness and knitting characteristics, cooked curd size, 19 final curd texture, protein breakdown, body, texture and flavor. In addition, bitterness did not develop when excess rennet was added. Research by Edelsten and Jensen (1970) revealed that M. miehei rennet contained at least three different coagulation-active components, each with its own character- istic temperature dependence. Sternberg (1971) also indicated the presence of more than one protease in M. miehei. Comparison of Fungal Proteases Birkkjaer and Thomsen (1970) manufactured Samsoe and Danho cheese with "Noury-Rennet" (M. pusillus) and "Suparen" (M. parasitica) using "Hansens Fifty-Fifty" animal rennet (50% calf rennet and 50% pepsin) as the control. Both of the microbial rennets took 10-15 minutes longer to form a gel than did the animal rennet. Cheese of good quality could be manufactured with the M. pusillus rennet providing calcium chloride was added but the M. parasitica rennet produced a cheese described as mealy, hard, bitter, acid, off flavor and short in body. Edwards (1969) manufactured Cheddar cheese with various milk coagulants and found E. parasitica rennet and animal rennet to produce bitter flavors but cheese manu- factured with M. pusillus protease did not suffer from this flavor defect. Casein was implicated as the source of the bitter peptides. 20 In a study by Mickelsen and Fish (1970) the pro— tease from M. pusillus Lindt and E. parasitica both showed greater proteolytic activity than did veal rennet or pepsin on whole, as- and B-casein. Of the two fungal rennets, the extract from E. parasitica was more proteolytic on the casein preparations. In general, the fungal rennets produced more NPN when incubated in casein sols and more soluble nitrogen when mixed with cheese paste than did calf rennet or pepsin. Electrophoretic patterns of major casein fractions subjected to the proteolytic action of the enzymes, revealed considerable more proteolysis by the fungal rennets. Labuschagne and Jaarsma (1970) manufactured Gouda and Cheddar cheese using E. parasitica, M. miehei and two strains of M. pusillus. Results showed the microbial rennets were an acceptable substitute for calf rennet providing slight variations were made in the manufacturing procedure. The milk clotting activity of the microbial rennets was influenced by variations such as ionizable calcium, acidity, temperature of the milk and conditions of pasteurization. These variables were observed to affect animal rennet to a lesser extent. The microbial rennets tended to produce a soft curd and the problem was counter- acted by adding calcium chloride to the milk and ripening to slightly higher acidity. Firmer curd could not be obtained by adding more fungal rennet. There was a tendency for the cheese made with microbial rennet to have 21 higher moisture content. In addition, fat losses in the whey were higher with Cheddar cheese. Edwards and Kosikowski (1969) manufactured Cheddar cheese using commercially available microbial rennets obtained from the organisms, M. parasitica, M. pusillus and M. miehei. Electrophoretic patterns showed that calf rennet attacked mainly a—casein; E. parasitica acted mainly on B-casein while the two Mucors hydrolyzed both as- and B-casein to the same degree. In addition these workers noted that bitter cheese was produced with calf rennet and the Endothia rennet but not with the Mucor rennets. Vanderpoorten and Weckx (1972) compared the same three commercial fungal rennets with respect to their affect on casein, casein components and casein in cheese. Generally the microbial rennets liberated more NPN from whole casein, as- and B-fractions, than did veal rennet. E. parasitica was the most proteolytic towards all casein fractions except for K-casein. In this instance, calf rennet and the Mucor rennets displayed stronger activity when the release of NPN was followed over a period of two hr. Electrophoretic analysis of casein digests revealed that as- and B-casein produced a characteristic pattern for each coagulating agent. Gel patterns indicated that E. parasitica was the most proteolytic except in the case in K—casein. M. pusillus Lindt and M. miehei exhibited the greatest proteolytic activity on K-casein. 22 By electrophoresis of 4 week old Gouda cheese, Vanderpoorten and Weckx (1972) revealed that microbial rennets produce gel patterns which can be clearly differ- entiated from the electropherogram of Gouda cheese made with veal rennet. All three fungal rennets produced an additional band ahead of the aS-casein that was not evident when calf rennet was used. M. parasitica produced an extra band behind the B-fraction which could not be demonstrated when the Mucors and calf rennet were used as coagulants. This phenomenon was also observed by other workers (Mickelsen and Fish, 1970; Edwards and Kosikowski, 1969). Thomasow (1971b) observed that all three commercial fungal proteases and animal rennet attacked K-casein. The remaining caseins were not attacked by animal rennet to any extent but the fungal proteases had a greater affect on the as- and B-caseins. This investigator also observed that pH variation in the milk had the least affect on M. parasitica while pepsin-calf rennet mixtures were most strongly influenced. Tam and Whitaker (1972) compared the rates and extent of hydrolysis of aS-, B-, and K— and whole casein at pH 3.0, 3.5, 5.5 and 6.0 by crystallized chymosin, crystallized pepsin and purified proteases of M. pusillus and E. parasitica. The results obtained can be summarized as follows: 23 a. E. parasitica protease proved to be more active than the other three enzymes on all substrates at each pH assayed. b. With as-, K- and whole casein, the initial rates and extent of hydrolysis tended to decrease as the pH was lowered from 6.0 to 3.0 c. All enzyme preparations hydrolyzed B-casein more extensively at pH 3.5 than at 3.0. d. At pH 6.0, all four enzymes hydrolyzed K—casein the most rapidly. This was followed in turn by a- and B-casein. e. The initial rates of hydrolysis of B-casein at pH 6.0 was slow by all enzyme preparations. f. B-casein was hydrolyzed more rapidly at pH 5.5 than at 6.0 by all of the coagulating enzymes. Bitterness Associated With Calf Rennet Substitutes In the preceding section, the data reported by several authors indicates that bitterness is often associ- ated with fungal proteases while others contend that such problems do not exist. Kikuchi and Toyoda (1970) manufactured Edam, Gouda and Cheddar type cheeses from M. pusillus Lindt and Bacillus polymyxa. Curd produced by the microbial rennets was softer and shattered more easily at cutting when com- pared to calf rennet. A bitter taste was frequently found in cheese made with microbial rennets, especially in 24 cheeses made with crystalline enzymes. These authors concluded that the bitter taste in cheeses made with microbial rennets was not caused by the action of contami- nant proteases but was an inherent characteristic of the primary enzymes. In contrast to Kikuchi and Toyoda's theory (1970) Organon (1971) indicates that non-specific enzymes present in the microbial rennets of Mucor, Endothia, Rhizopus, Monascus and Colletotrichum are responsible for bitter flavor. The non-specific enzymes can be removed by adsorption to silicate. The ratio of coagulating activity to proteolytic activity can be increased from a range of 6,000-ll,000, to a range of 14,000-20,000 following such treatment. Richardson and Nelson (1968) reported that fungal milk clotting enzymes produced more bitterness in cheese paste than did rennet or pepsin. Dulley and Kitchen (1972) demonstrated that bitterness can also be produced by calf rennet and is due to the release of simple peptides rather than phosphopeptides as implied by earlier workers. Despite problems with bitterness, Kikuchi and Toyoda (1970) indicate that microbial rennets could be used to replace the conventional calf rennet in cheese making if certain aspects of manufacture were modified. These included setting temperature, cutting time and cooking method. Optimizing these factors would probably 25 have a greater affect on curd characteristics rather than actually eliminating the bitterness problem. Potential Calf Rennet Substitutes Bacterial Rennets Bacillus Species.--A bacterial protease produced by M. polymyxa has also received some attention as a calf rennet substitute. Itoh and Thomasow (1971) noted that milk clotting enzyme hydrolyzed casein fractions (as, B and K) extensively at pH 6.5. Proteolysis was not specific but continued as general proteolysis. This was reflected in NPN as well as electrophoretic patterns of each of the major casein components. This bacterial protease has a pH Optimum of 8.0 and its proteolytic activity on casein decreases markedly below pH 7.0. Itoh and Thomasow (1971) concluded that application of this protease to cheese making would require proper pH control to prevent over- proteolytic action. If proteolysis of as- and B-casein were extensive, bitterness was encountered. Thomasow (1971b) studied the milk clotting activity of E. polymyxa and Bacillus subtilus in addition to extracts from several fungi. The bacterial proteases exhibited very strong proteolytic activity which resulted in a weak curd. Extensive protein breakdown caused off- flavor and poor consistency in the cheese. 26 Dutta £3 31. (1971) examined the protease from g. subtilus a spore forming bacterium. The milk clotting activity (MCA) of this enzyme was affected greatly by the concentration of calcium chloride with the pH optimum for clotting and proteolytic activity being 6.0 and 8.0 respectively. No conclusions were drawn as to the acceptability of this rennet as a calf rennet substitute. Stefanowa—Kondratenko gt 31. (1971) prepared a rennet extract from Bacillus mesentericus (strain 76) and studied its suitability for manfacturing Bulgarian cheese from sheep and cows' milk. Results showed the keeping quality and taste of the experimental cheese to be equal to those of conventionally manufactured control cheese. Melachouris and Tuckey (1968) isolated a milk- clotting enzyme from Bacillus cereus. The clotting activity of this microbial rennet was less sensitive to pH changes of the substrate than calf rennet. This enzyme resembled calf rennet with respect to optimum temperature for clotting and inactivation. The microbial rennet was more proteolytic than calf rennet and degraded casein fractions continuously and non-specifically with B-casein being the most susceptible to hydrolysis. The action of this enzyme on K-casein was similar to that obtained with calf rennet. 27 Fungal Rennets Aspergillus niger.--Osman et al. (1969a, 1969b) studied a number of fungi and found that M. BEESE (Isolate no. 58) produced extracellular proteases, one of which had high MCA and low proteolytic activity. The course of proteolysis in the first stage of enzymatic action was similar to that of calf rennet. Basidomycetes.--Kawai and Mukai (1970) and Kawai (1970a, 1970b) surveyed the milk clotting enzyme produced by a large number of Basidiomycetes. Research revealed that rennet extract from two strains, £5225 lacteus (Fr.) and Fomitopsis penicila (Fr.) Karst, could be employed to produce Cheddar cheese of good quality. Rennet of the latter type produced a slightly bitter taste after five months. Like many other microbial proteases, these acid proteases are also affected by (Ca++) ion concentration. l. lacteus had MCA to proteolytic activity ratio which resembled the mucor rennet. Kawai (1970a) indicates the protease of 3. lacteus is the most promising Basidiomycete substitute for calf rennet. Plant Rennets Papain.--Balls and Hoover (1937) studied the milk clotting action of papain but did not describe its use as a rennin substitute. Dewane (1960, citing Nasher) points 28 out that when papain is used alone it is not a satisfactory calf rennet substitute. EigiM.--Whitaker (1959) suggested that Ficin, a sulfhydryl protease similar to papain, could be used in cheese making. In an earlier paper, Krisnamurti and Subrahmanyan (1949) reported that in cheese making trials ficin compared favorably to rennet as a coagulant in that cheeses of equal quality were produced. Ficin is more versatile than calf rennet as it will clot not only animal milk, but also soya milk. Cardoon.--Other vegetable proteases used include rennet extracts from the flowers of Cardoon (Cynara cardundulus). This extract was traditionally used by farmers of Portugal in making Serra cheese according to Sé and Barbosa (1970a). Cardoon extract was regarded as a satisfactory substitute for animal rennet and was con- sidered more suitable than calf rennet for coagulating sheep milk. In a related study, 85 and Barbosa (1970b) observed that Cardoon rennet showed higher proteolytic activity than calf rennet and did present some technological problems in Edam cheese making. However, it was a satisfactory clotting enzyme for soft bodied cheeses like Serra and Roquefort although there was some loss in yield with the latter. Sé and Barbosa (1970c) concluded that Cardoon 29 extract and animal rennet behaved almost identically in both cows' and sheep milk. Microbial Rennets in General Behnke (1967) surveyed 20 different rennet prepa- rations of animal and microbial origin. The microbial proteases exhibited high rennet strength and were less dependent upon pH than were calf rennet and porcine pepsin. Generally, microbial proteases have 10 to 100 times the proteolytic activity of pepsin and rennin. Some microbial proteases did not show any primary reactions despite normal rennet strength. Srinivasan 33 El: (1970), without specifying the source of microbial rennet, produced good quality soft varieties of cheese from both cows' milk and buffalos' milk. The body, texture and flavor of the cheese produced was found to depend more on the source of the milk than on the type of rennet used. Kyla-Siurola and Antila (1970) also compared microbial rennets to calf rennet using the normal manu- facturing method for Finnish Edam and Emmental cheese. No differences were Observed between acidity, dry matter, fat and total nitrogen. In addition, the free amino acid content was about the same and differences in soluble nitrogen at the end of six weeks tended to diminish with continuing proteolysis. There were no differences in the 30 electrophoretic patterns of cheeses made with animal rennet and the microbial rennets. Additional Calf-Rennet Substitutes The vast literature on this subject precludes the practicality of presenting an exhaustive review of calf rennet substitutes. This review has attempted rather to describe the major proteases that are, or have the potential of being, acceptable calf rennet substitutes. Hundreds of other sources of calf rennet substi- tutes have been sought and are described or cited by various authors including: Matsubara and Feder (1971), Babbar gt 31. (1965), Dewane (1960), Labuschagne and Jaarsma (1970), Veringa (1961), Behnke (1967), Sardinas (1968), Kawai and Mukai (1970), Osman gt 31. (1969a), Arima and Tamura (1967), Abel-Fattah 33 31. (1972), Sannabadth 23 31. (1970), Knight (1966), Arima eE g1. (1970), Genin (1968), Oruntaeva and Seitov (1971), Chaudhari and Richardson (1971). EXPERIMENTAL PROCEDURE 8 Microbiological Techniques Selection of Cultures Two strains of E. rggueforti were obtained from American Type Culture Collection (ATCC); 6987, 10110. A third strain was provided by Snow Brand Milk Products (Tokyo, Japan) and was designated BP-l3 by Niki et‘al. (1966). All cultures were carried on Czapek—Dox agar con- taining 0.75% (w/v) Proteose-Peptone no. 3 (Difco) and 0.75% (w/v) sodium caseinate. Detection of Proteolytic Activity Four types of media, poured into disposable plastic petri dishes, were used to qualitatively detect proteolysis. Type I: This was an improved medium used by Martley 33 E1. [(1970), (Appendix)] to detect proteolysis by a wide variety of organisms. The sodium caseinate was dissolved in 300 ml of 0.015 M sodium citrate and added to the Standard Methods Agar (SMA) which had been hydrated in 700 ml of the same buffer. The medium was autoclaved at 121 C for 15 min and 31 32 just before pouring into sterile petri dishes, 20.0 ml of sterile l M calcium chloride was added to a liter of the liquid medium. Type II: The composition of this medium was the same as Type I except the casein was sterilized using hydrogen peroxide and heat. Ten 9 of sodium caseinate was dissolved in 300 ml of water containing 4.41 g of trisodium citrate. To the casein solution, 0.4% hydrogen peroxide was added (v/w) and heated to 55 C for 15 min. The solution was then cooled to 30 C and 0.5 m1 of sterile catalase (Nutritional Biochemical Corporation) was added and the solution allowed to stand at room temperature for 8 hr. Residual hydrogen peroxide was assayed by adding saturated potassium iodide solution. The SMA (obtained from BBL), calcium chloride and water were mixed in the same proportion as in the Type I medium, sterilized, cooled to 50 C, then mixed with the sterile casein solution. Sterility of the casein solution was determined by Standard Plate Count. Type III: Thirty-five g of Czapek-Dox Broth (Difco) was dissolved in 700 ml of water (Appendix). To this, 15.0 g of agar was added and autoclaved to obtain sterility. The casein was prepared as with the Type II medium. Type IV: The composition of this medium, devised by Meyers and Knight (1958), is listed in the Appendix. After formulation of the medium, 1.5% (w/v) agar was added. The solution was autoclaved, cooled and then mixed 33 with the peroxide sterilized casein solution as previously described. Prior to pouring, 20 m1 of sterile 1 M calcium chloride solution was added. The plates, containing Types I through IV media, were held at 30 C for 48 hr and stored at 4 C until required for use. Proteolytic activity by each of the three strains of g. roqueforti was detected by streaking 0.1 m1 of a standardized spore count (in a 0.01% sterile soap solution) on the surface of each type of agar medium and incubating at 25 C for three to four days. Standard Plate Counts Standard plate counts were determined as described by the American Public Health Association (1960). Spore Counting Penicillium spores were removed from Czapek-Dox agar slants by adding 100 m1 of a 0.01% (w/v) sterile soap solution and shaking gently. An estimate of spore count/ml was made by means of a Spencer bright line hemacytometer. Shake-culturing Culturing was carried out at 225 rpm on a gyrotatory shaker (New Brunswick Scientific) held at 25 to 27 C for 72-78 hr. One £ erlenmeyer flasks were used for shake- culturing. The broth consisted of 35 g of Czapek-Dox broth and 5.0 g of Protease-Peptone no. 3 per i of solution. Prior to sterilization at 121 C for 15 min, the pH of the 34 broth was adjusted to 4.0 with 1 N hydrochloric acid. Three hundred m1 of broth was added to each flask. Following sterilization, an inoculum of 90 to 100 x 106 spores was placed in each flask. Preparatigp of Cell Free Extract (CFE) The 3' roqueforti (BP-l3), which had been shake- cultured at 225 rpm for 72-78 hr, was added to 250 ml polycarbonate flasks and centrifuged in a swinging bucket type head (International Model K) at 1000 G for 20 min. The spores and mycelial mass compacted at the bottom of the flasks allowing the supernatant to be decanted easily. The CFE was prepared by passing the above supernatant through two Millipore filters, 0.8 and 0.45 u, then storing in sterilized flasks until needed at 4 C. Preparative Procedures Buffers Universal Buffer.--To obtain effective buffering capacity over a wide pH range, requires a multi-component buffer with pK values approximately 2 pH units apart. Coch Frugoni (1957) describes the preparation of a Universal buffer of constant ionic strength covering the pH range of 2 to 12 at integral pH units. This buffer is 0.04 M with respect to phosphoric, acetic and boric acid (Appendix). 35 Additional Buffers.--Citrate and other buffers were prepared as described by Dutta and Grzybowsk (1961). Casein Sodium Caseinate.--Casein was precipitated from freshly separated skimmilk (0.04% butterfat) at pH 4.6, with 1 N hydrochloric acid and 30 C. After washing the precipitate with copious amounts of distilled water, the protein was redissolved by adding 1 N sodium hydroxide to bring the pH of the suspension back to 7.0. The precipi- tation and washing process were repeated. The casein was then freeze dried to a final platen temperature of 33 C by means of a Virtis model 42 freeze drier. Hammersten Casein.--The method of Dunn (1949) was followed for the preparation of Hammersten casein. Sodium caseinate was prepared as previously described with the final casein precipitate being suspended in 95% ethanol. Following a series of ethanol and ether washes to remove moisture and fat, the purified product was held at room temperature for 8 hr to remove the ether and attain moisture equilibrium. The final product contained 6.73% moisture as determined by the vacuum oven method (A.O.A.C., 1960). Hammersten casein was used in all assays for protease activity. 36 Enzyme Substrate.--A 1% casein sol was prepared by dissolving 10.72 g of Hammersten casein in 800 m1 of 0.03 M citrate buffer at pH 8.1. The suspension was placed in a boiling water bath for 15 min, cooled, pH adjusted to 5.75 with 1 N hydrochloric acid and ionic strength brought up to 0.3 with sodium chloride. Following dilution to 1 K with 0.03 M citrate buffer (pH 5.75, u=0.3) the suspension was filtered through a 0.4SUm Millipore filter to remove micro- organisms and insoluble material. The 1% casein sol was stored in a sterile flask at 4 C. Dialysis Tubing The method of McPhie (1971) was used to prepare the dialysis tubing. Approximately 50 feet of 2.5 cm tubing was placed in 2 Z of 50% ethanol and simmered for 1 hr. The ethanol was drained and this treatment repeated. This was followed by two repeated immersions, 1 hr each, in 10 mM sodium bicarbonate. After being submerged in 1 mM EDTA for another hr, the tubing was rinsed in two changes of distilled water for 1 hr each. The tubing was stored at 4 C in distilled water containing 0.02% sodium azide (w/v). Dialysis Samples requiring dialysis were placed in 4 2 of deionized distilled water and stirred continuously at O C by means of a magnetic stirrer. 37 Pervaporation 8015 in dialysis tubing were pervaporated at room temperature by means of a fan placed 12 to 16 inches from the sample being concentrated. Lyophilization Approximately 15 to 30 m1 of sample was placed in a 50 ml round bottom flask fitted with a 35/25 ball socket. The flask was then attached to a Rinco evaporator by means of an (24/40 I - 35/25 ball) adaptor and shell frozen in a dry ice-acetone bath. When solidified, the samples were connected to a glass "udder-type" lyophilizer (Kontes) by means of another adaptor (34/45 $ - 35/25 ball). Vacuum was attained with a'Cenco Hyvac 14 pump. A cold finger containing dry ice and acetone was used to trap moisture sublimed from the frozen samples. After 12 to 16 hr the lyophilized samples were removed and stored at 4 C in a desiccator. Standardized Assay Procedure for Proteolytic Activity The Lowry modification of the Folin reagent was used for determining proteinase activity (McDonald and Chen, 1965). This modified procedure incorporates a pretreatment with alkaline copper sulfate. Substances that 'give a positive biuret reaction also produce color with the I?olin reagent. Without the COpper treatment, only substances (zontaining tyrosine and tryptophane produce color when the 38 Folin-Ciocalteau reagent is added, according to Herriott (1941). Assay Procedure To measure proteolysis, 0.5 ml of the proper dilution of enzyme was added to 2.0 m1 of a 1% sol of Hammersten casein incubated at 30 C for 9 min. The reaction was terminated by the addition of 3.0 ml of 6% trichloroacetic acid (TCA). Blank determinations were made by adding TCA to the casein substrate, mixing, then adding the enzyme solution. After standing 20 min the sol was filtered through Whatman no. 44 filter paper. One-half ml of filtrate was then mixed with 5 m1 of alkaline copper sulfate-sodium tartrate solution (Appendix). The solution was allowed to stand 10 min at room temperature. .One-half m1 of l N Folin-Ciocalteau reagent was added, and the solution was mixed within 2 sec on a Fisher mini-shaker. The pH of the solution was 9.9 to 10.1 which is the optimum for color development according to McDonald and Chen (1965). The color was developed at room temperature for 1 hr or at 28 C for 30 min. Absorbance was read at 600 or 750 nm depending on color intensity. Casein Standard Curves Two standard curves were prepared from Hammersten czasein using the Lowry-Folin procedure for color develop- rnent. From these two curves it was determined that éibsorbances in the range of 0.0 to 0.19 (0 to 50 ug casein) 39 and 0.16 to 0.45 (50 to 200 pg casein) would be read at 750 and 600 nm respectively when determining proteinase activity. The two standard curves for casein were prepared by peptizing Hammersten casein in 0.03 M citrate buffer and selecting appropriate alliquots to cover the range desired. To 0.5 ml of the casein sol, 2.0 ml of alkaline copper sulfate-sodium tartrate solution (Appendix) were added with the alkali being 0.2 N sodium hydroxide in 2% sodium bicarbonate. Color was developed as described in the "assay procedure." Bovine Sepgm Albumin (BSA) Standard Curves Two standard curves were prepared in the same manner as described for casein. Absorbances in the range of 0.0 to 0.215 (0 to 50 ug of BSA) and 0.160 to 0.570 (50 to 200 pg BSA) were read at 750 and 600 nm respectively. Enzyme Unit One unit of enzymatic activity was defined as that amount of enzyme which would produce a Lowry-Folin absorbance increase of 0.01/min when read at 600 nm or AA of 0.012/min at 750 nm when using a 1% sol of Hammersten casein at pH 5.75 as substrate. 40 Determination of Variables Involved in Assay Optimumng Activity was measured at various pH levels using sols of 1% BSA and Hammersten casein dispersed in Universal buffer. Assays were conducted as previously described. Stabilitygtong In order to determine the stability of the BP-13 protease to pH, enzyme sols were adjusted to integral pH units from 1 to 12 by means of hydrochloric acid or sodium hydroxide. The enzyme sols were held 48 hr at 4 C, the pH adjusted to 5.75 and assayed for residual activity. Optimum Temperature The relative activity of the BP-l3 protease was determined at 20, 23, 25, 30, 35, 40, 45, 50 and 60 C. This was a nine min end point assay. In a related experi- ment, activity was assayed at 3, 6 and 9 min to determine the rate Of hydrolysis at temperatures of 20, 25, 30, 35, 40, 46, 48, 50 and 55 C. Stability to Temperature The protease was incubated at temperatures of 25.0, 30.0, 35.0, 40.0, 45.0, 50.1, 53.2, 56.0 and 59.9 C. For temperatures up to and including 45 C, 0.5 ml samples of enzyme were taken at 10, 20 and 30 min. At temperatures greater than 45 C, samples were removed at 3, 6 and 9 min due to rapid inactivation of the enzyme. In each case 41 the enzyme was incubated at the above temperatures at pH 5.0 in citrate buffer (0.03M, u = 0.3). After exposure to various temperatures for the required time interval, 0.5 ml of the enzyme sol was pipetted into test tubes packed in ice. For purposes of assaying for activity, 2 ml of 1% casein (pH 5.75) at 30 C were added to 0.5 ml aliquots of enzyme sol incubated at the same temperature. Effect of Substrate Concentration The rate of hydrolysis of two casein sols, 0.5 and 1.0% casein (pH 5.75), were followed over a period of 30 min with the TCA soluble nitrogen being determined by the Lowry-Folin procedure at time intervals of 3, 6, 9, 15, 20, 25 and 30 min. Hydrolysis of a 0.5% casein sol (pH 4.0) was also studied in a similar manner. The casein was dispersed in 0.04 M Universal buffer (u = 0.3). Study of Autolysis An enzyme sol collected from G50/GlOO Sephadex columns was incubated at 30 C with samples being taken at 2, 18, and 30 hr. Each aliquot was then analyzed for TCA soluble nitrogen, protein and activity. Purification of BP-l3 Protease Precipitation of Enzyme Solvents.--Ethanol, methanol and acetone were added to 50 m1 of CFE until solvent concentrations of 60, 42 70, 80 and 90% (v/v) were Obtained. After 12 hr of storage at 4 C the suspensions were centrifuged at 30,000 G for 30 min in a refrigerated International centrifuge (Model HR-l). The supernatant was decanted off and the precipitate dissolved in Universal buffer at pH 5.75. Both fractions were assayed for activity. Salt.--Ammonium sulfate was added to 50 m1 of CFE to obtain final saturations of 40, 50, 60, 70, 75, 80, 85 and 90% at 25 C. The samples were refrigerated (4 C) over- night and then centrifuged and assayed as described in the preceding paragraph. Ultrafiltration The CFE was concentrated in an Amicon thin channel filtration system (TCF-lO). The membrane (UM 10) was 90 mm in diameter with an apparent pore diameter of 1.5 x 10"3 um. The manufacturer indicates the membrane has a cut off of 10,000 Daltons when calibrated with polyethylene glycol. Ultrafiltration was conducted under 40 psi of nitrogen pressure in a cold room held at 0 C. Approximately 1000 ml of CFE were concentrated to 50 ml. Gel Filtration Preparation of Sephadex.--G50 fine and G100 Sephadex were hydrated in 80 to 90 C citrate buffer (0.04M, pH 5.0, u=0.3). The beads were allowed to settle five min, then the supernatant and fines were siphoned off. This process 43 was repeated. Following deaeration of the Sephadex scilution, the beads were allowed to hydrate for 12 hr. The K25/45 and K16/100 columns (Pharmacia) were poured as described by Fischer (1971) . Application of Sample.--Fifteen ml of concentrated CFE was applied to the top of the 650 Sephadex column by means of a 10 m1 syringe. After all the concentrate had entered the gel, a small amount of buffer was used to elute the sample from the top of the column. The column was then filled with the same citrate buffer and connected to the eluate reservoir. A constant hydrostatic head pressure of 15 to 18 cm was maintained on both the G50 and G100 columns by means of a Mariotte flask. The flow rate was regulated to obtain 1 drop/12 sec or approximately 15 ml/hr. The partially purified enzyme obtained from the G50/6100 Sephadex was applied in a similar manner to the K16/100 (1.6 x 100 cm) column containing G100 Sephadex. Chromatography.--The G50 column was run downflow itho the top of the G100 Sephadex by means of a flow adaptor. An LV-4 valve (Pharmacia) was placed between the In“: columns so a major portion of the Proteose-Peptone fraction could be diverted from the G100 Sephadex column (Figure 15). Eluate from the 6100 Sephadex column passed through a flow regulating value, and was then monitored at 254 nm using an ISCO dual beam UA-2 analyzer. From the monitor, the effluent travelled through a flow interrupter 44 valve designed to momentarily stop the flow while the fraction collector changed tubes. Drops of effluent from the flow interrupter valve were recorded as they passed through the drop counter to the ISCO fraction collector (Model 326). After 75 drops (5 ml) the delay timer on the fraction collector was activated. This device compensated for the time the effluent took to travel from the photocell to the flow interrupter valve. This was set for 100 sec for the two K25/45 columns and 150 sec for the Kl6/100 column. When the delay timer reached 0 sec, the flow interrupter valve would close and the shuttles of the fraction collector advanced a new test tube into position. Each time tubes were changed, this event appeared as a dot on the left side of the recorder paper. Emoval of the Nucleic Acids Certain preparations of CFE contained excessive quantities of nucleic acids which tended to obstruct the G50 Sephadex column. Rhodes e_t_ _a_]_._. (1971) outlines a Procedure for removing nucleic acids. This method involves the use of 10% (v/v) of a 2% (w/v) solution of protamine SUIfate added to the CFE adjusted to pH 7.0. After stirring for 20 min the suspension was centrifuged at 15,000 G for 20 min. The supernatant was decanted off, concentrated by Ultrafiltration, then applied to the GSO Sephadex as Previously described. 45 Further Purification of BP-13 Protease Additional fractionation procedures were used in an attempt to remove small molecular weight components from the purified BP—13 protease. Ion Exchange Several types of ion exchangers were employed: DEAE, SE and P cellulose (Bio-Rad); IRC-SO and IR-120 Amberlite (Rohm and Haas). The functional group, pK and method of regeneration for each type of ion exchange are listed in Table 8. Preparation of Celluloses.--DEAE cellulose was hydrated in l M sodium hydroxide for 30 min at room temperature. This was followed by 500 m1 washes of 1.0 M sodium hydroxide, water, 0.5 M hydrochloric acid, water and 1.0 M sodium hydroxide. The SE and P celluloses did not require this pretreatment. One gram of each ion exchanger was stirred in a 80 ml of buffer. After settling 15 min, the cloudy supernatant was discarded. Forty m1 of the same Universal buffer were added, stirred and treated as before. Two additional repetitions of this experiment prepared the ion exchanger for use. Preparation of Resins.--Amberlite IRC-50 and IR—120 were prepared for use following the procedure of Bailey (1967). This involves a series of washes with hydrochloric 46 acid or sodium hydroxide of various strengths, water, and finally buffer for equilibration. Preliminary Ion Exchange Experiments.--Pilot studies were performed in order to determine the optimum pH for absorption of the enzyme to the ion exchanger. This entailed mixing enzyme and ion exchanger at several pH levels in Universal buffer and then determining the residual activity in the supernatant. Preliminary experiments were carried out with only the DEAE and SE cellulose at integral pH units of 3 to 7 and 3 to 6 respectively. Twenty ml of Universal buffer (u = 0.1), 1.0 g of ion exchanger, and approximately 100 units of purified enzyme were mixed and centrifuged. The supernatant was then assayed for activity. Ion Exchange Experiments.--Each of the ion ex- changers was used under varying conditions of pH and/or ionic strength (Table 10) in an attempt to further purify the BP-13 protease. The cellulosic exchange resins were packed in 0.9 x 15 cm columns (K15/30, Pharmacia) to a height of 8 to 10 cm. A flow rate of 5 to 10 ml/hr was established for all columns. pH and ionic strength gradients were prepared by means of a GM-l gradient former (Pharmacia). 47 Electrodialysis Electrodialysis equipment was fabricated from a 1 gal Nalgene container, Plexiglass, nylon bolts and stainless steel (Figure 25). The bottom end of the dialysis tubing (7.5 cm, flat width) was doubled back 2 cm, then placed on the 1/4 inch Plexiglass frame. The nylon bolts were tightened down and the frame was then inserted into the slotted Plexiglass head fitted on top of the Nalgene container. Two stainless steel electrodes (0.065 inches in diameter) were centered 1.5 inches from the dialysis tubing. Protein sols were injected into the cellulose tubing through a slot in the upper portion of the Plexiglass frame. The electrodes were connected to a Heathkit high voltage power supply (Model 1P-32) with a maximum potential of 400 V at 150 ma. The surface of the dialysis tubing was continuously flushed with deionized distilled water obtained from a 20 2 Mariotte flask. Following electrodialysis the enzyme was freeze dried. Analytical Procedures Protein Lowry-Folin.--Protein was determined in the same manner as described for the preparation of the standard curves. An absorbance of 0.214 at 750 nm was equivalent to 51 pg of purified BP-13 protease as determined by the Kjeldahl procedure. 48 Kjeldahl.--From 5 to 50 mg Of protein were mixed with 4.0 m1 of digestion mixture (Appendix), heated for 2 hr, then cooled. After the addition of 1 ml of 30% hydrogen peroxide the digestion was continued for 2 hr. Upon cooling, the sides of the flasks were rinsed down with distilled water. The digestion mixture was then neutralized with 20 m1 of 40% NaOH and approximately 35 ml of distillate was collected in 15 ml of 4% (w/v) boric acid. This solution was then titrated with 0.060 N HCl to the same pH as the blank. Recoveries of nitrogen were checked with DL tryptophan which had been desiccated over phosphorus pentoxide for one month. This method for nitrogen determination is a modified AOAC procedure (1960). Carbohydrate Carbohydrate was qualitatively determined by the Molish test (Clark, 1964). This consists of adding 0.5 ml of a 0.5% (w/v) protein sol of BP-13 protease to 0.1 ml of 5% alcoholic a napthol and mixing. Without agitation, 1 m1 of concentrated sulfuric acid was layered beneath the aqueous solution. Glucose was used as a control in this experiment. Acrylamide Gel Electrophoresis All electrophoretic analyses were made using an EC 470 vertical gel electrophoretic cell (E.C. Apparatus) equipped with a buffer pump to circulate electrode buffer from the lower to upper chamber (Jordan and Raymond, 1967). 49 Alkaline Gels.--Alkaline gels were prepared and protein samples electrophoresed by the method of Melachouris (1969). This is a discontinuous system in which the 8% (w/v) running gel (Cyanogum 41, E.C. Apparatus) was dissolved in 0.38M Tris-HCl buffer at pH 8.9 while the 5% spacer gel (w/v) utilized 0.062 M Tris-HCl buffer at pH 6.7. After adding 0.1% (v/v) N N N'N' Tetramethylethylene- diamine (TEMED) the gel solution was filtered through Whatman no. 44 filter paper and stored at 4 C until required for use. Gels were warmed to 25 C and polymerized with ammonium persulfate; 0.5 ml of a 2% solution (w/v) per 50 ml of gel. Spacer and running gels were poured using the procedure described by Jordan and Raymond (1967). This method consists of occluding the bottom of the column with running gel by placing the cell at an angle of 45° while 50 ml of running gel polymerized. Excess buffer was removed from the surface of the blocking gel by means of a sponge strip. This was followed by the addition of running gel which is allowed to polymerize with the cell in the vertical position. Again the excess buffer was removed from the surface of the running gel. The electrophesis cell was then placed in the horizontal position, slot former inserted, and chamber filled with spacer gel. Following polymerization, the excess spacer gel was removed. After the addition of 2 R of electrode buffer to both the upper and lower chambers, the 8 place teflon slot former was 50 removed and samples applied by means of a 50 pl syringe. Sample size ranged from 15 to 20 pl of a 5% protein sol dissolved in spacer gel buffer diluted 1:1. When urea was not employed, 5% sucrose was added to increase the specific gravity of the samples. Bromphenol Blue (J. T. Baker) was also added as a marker to each protein suspension. Electrophoresis was performed at 225 v for 2.5 to 3.0 hr. Alkaline gels were also run utilizing 6M urea in both running and spacer gels. When 6M urea was added to the protein samples, sucrose was omitted. Acid Gels.--A discontinuous system described by Jordan and Raymond (1970) utilizes 0.2 M tris-citric acid in 12% (w/v) acrylamide gel and 0.37 M glycine-citric acid in the electrode chamber. Twelve percent acrylamide was found to be too high and this was reduced to 8% in all acid gels. The catalyst system is composed of 0.7% ascorbic acid, 0.0025% ferrous sulfate with 0.03% hydrogen peroxide (30%) added immediately prior to pouring. Coolant was circulated through the cell to remove the heat of polymeri- zation; otherwise the gel would contract on cooling, leaving air spaces between the gel slab and cooling plates. A spacer gel was not poured. Basic Fuschin (Sigma) was used as a marker. Electrophoresis was conducted at 225 v for 3.0 to 3.5 hr. 51 Sodium Dodecyl Sulfate (SDS) Gel Electrophoresis for Determination of Molecular Weight.--Five percent acrylamide gels containing 0.1% SDS (Pfaltz and Bauer Inc.) were used for the determination of molecular weight. This is a continuous buffer system which utilizes 0.1 M phosphate buffer at pH 7.1 (Shapiro, 1967). Protein samples were dispersed in phosphate buffer containing 1% SDS and denatured by heating to 37 C for 3 hr. A short prerun of 15 min was made prior to introducing protein samples into the slots. Electrophoresis was performed at 75 v (95 ma) for 12 hr. Protein standards included aldolase, ovalalbumin, chymotrypsinogen A, ribonuclease A (Pharmacia) and pepsin (Calibiochem). Staining andDestainingof Acpylamide Gels Amido Black lOB.--Gels prepared by the method of Melachouris (1969) were stained for 8 min in a solution of 0.25% Amino Black 108 (E.C. Apparatus). The dye was dissolved in methanol, water and glacial acetic acid (5:5:1). The same acrylamide gels were destained electro- phoretically in 7% (v/v) acetic acid. Coomassie Blue.--Prior to staining with Coomassie Blue, the gels were fixed for 1 hr in a solution of 15% TCA. Staining required 6 to 8 hr immersion in a 1% 52 aqueous solution (w/v) of Coomassie Blue diluted 1:10 with 15% TCA. The excess stain was removed by soaking in 15% TCA for 12 to 20 hr. Both acid and SDS gels were stained and destained by this procedure. Absorption Spectra Enzyme and nucleic acid-protein peaks were scanned from 300 nm down to approximately 240 nm with a Beckman scanning spectrophotometer (ACTA). The peaks were scanned at the rate of 0.1 nm/sec with the recorder set at 20 nm/ inch. Analysis of Insulin (B Chain) Hydrolysates Hydrolysis.--Ten mg of oxidized B chain of Insulin (Mann Research Laboratories) was dissolved in 1.0 m1 of 0.1 M ammonium acetate buffer, pH 7.2. When all the substrate was in solution, the pH was lowered to 4.25 with 0.5 N acetic acid. After the addition of 100 pg of enzyme, the solution was incubated at 30 C for 20 hr. Following centrifugation to remove insoluble material, the solution was concentrated to approximately 0.25 ml. High Voltage Electrophoresis.--Forty to 50 pl of concentrated hydrolysate was spotted on Whatman 3 MM paper (46 x 57 cm) and dried by a stream of hot air. The entire isolid support, except for the area dried, was dipped in a 53 pyridine, acetic acid and water (0.1:l.0:109) solution at pH 3.6. The paper was removed and dried to approximately twice the original weight. The same buffer system was used in the electrophoretic run, with varsol being used as the coolant. Electrophoresis was carried out at 2500 v (approximately 150 ma) for 1 hr on a High Voltage Electro- phorator, Model D (Gilson Medical Electronics). Descending Paper Chromatography.--After drying the electropherogram at 80 to 90 C, descending chromatography was conducted at 90° to the previous run. The mobile phase consisted of butanol, acetic acid and water (4:1:5). The three solvents were mixed together with the two phases being partitioned by means of a separatory funnel. The light phase was used in the descending trough while the heavy phase was placed in the lower portion of the chromato- graphic chamber. The chromatogram was allowed to develop for 15 to 18 hr, then removed and dried at 80 to 90 C. After spraying with ninhydrin reagent (Appendix) the Chromatogram was heated to 100 C in a closed cabinet, to activate the color reaction. Hydrolysis of Synthetic Substrates Nine substrates were peptides blocked on the N terminal end by a carbobenzoxy or acetyl group. Four peptides were not blocked on the N terminal end while two substrates were arginine esters. Hydrolysis of all substrates, except the two ester derivatives, was assayed 54 using ninhydrin. Each substrate was made up to a con- centration of 10 mM by dissolving in dilute NaOH and warming to 35 C. All substrates are listed in Table 11. Peptides.--The reaction mixture consisted of 0.5 m1 of substrate, 0.45 ml of 0.1 M citrate buffer at pH levels of 3, 4 and 5 (p = 0.4) and 0.05 ml of enzyme solution (1 pg/pl). Following incubation at 30 C for 1 hr, 2.0 ml of ninhydrin solution (Appendix) were added. Glass marbles were placed on the test tubes with color development being carried out at 100 C for 20 min. After cooling, 5.0 ml of 50% n-propanol were added. Absorbance was read at 520 nm in a Beckman DU-2 spectrophotometer. This is a modified method of Clark (1964). Esters.-- l. p-Tosyl arginine methyl ester (TAME) The BP-13 protease was assayed at pH levels of 3, 4 and 5 (Universal buffer) using a substrate concentration of 1.05 x 10‘3 M. One hundred pg of enzyme was added to 3.0 m1 of the substrate and the reaction followed over a period of 10 min at 25 C. Hydrolysis of TAME results in an increase in absorbance at 247 nm. 2. N-Benzoyl-L-Arginine ethyl ester (BAEE) Again the protease was assayed at the same pH levels as TAME using 1.0 x 10-2M BAEE. After incubation at 30 C for 1 hr the samples were titrated with 0.0498 N sodium hydroxide to the initial pH of each reaction mixture. 55 Determination Of Molecular Weight E§_§3I7511tration Molecular weight standards used for calibration of the 6100 Sephadex column (1.6 x 100 cm) included aldolase, ovalalbumin, chymotrypsinogen A and ribonuclease A with molecular weights of 158,000, 45,000, 25,000 and 13,700 respectively. Five mg of aldolase, chymotrypsinogen and 30 mg of sucrose were added to 1.5 m1 of 0.05 M phosphate buffer (pH 6.88, p = 0.4) mixed and allowed to stand for 10 min. After centrifugation at 1000 G for 10 min, 0.75 ml of the clear supernatent was added to the top of the G100 Sephadex. The two standards were eluted with the same phosphate buffer. A second solution consisting of ovalalbumin and ribonuclease A was treated in an identical manner. The void volume was determined using 0.5 m1 of a 0.5% Blue Dextran 2000 solution. Kav were calculated by: Kav = Ve - Vo / VT - Vo where Ve elution volume void volume VO V T total volume Enzyme Inhibitors §erine Protease Inhibitor The activity of trypsin (Mann Research Laboratories, 2x crystallized) porcine pepsin (Calbiochem, 3x crystallized) and purified BP-13 protease were assayed in the presence of 56 3 and 1.25 x 10.3 M concentrations of phenylmethyl 1.0 x 10' sulfonylfluoride (Calbiochem, B grade). The serine protease inhibitor was dissolved in 2-propanol then diluted 1:10. Trypsin was dispersed in Universal buffer (pH 8.0) con- taining 5% 2-propanol and 1.5 mM calcium chloride while pepsin and BP-13 protease were dispersed in the same solvent system buffered to pH levels of 2.5 and 3.0, respectively. Ten ml of each enzyme sol (60 pg/ml) was added to 10 ml of 3 and 2.5 x 10-3M, inhibitor at concentrations of 2.0 x 10- then incubated for 20 min at 25 C. The substrate (1% BSA) used for assaying proteolytic activity was adjusted to the same pH as the enzyme sol. When assays for activity were conducted above pH 4.0 the BSA was denatured by heating for 5 min in a boiling water bath. Activity was assayed as previously described. Sulfhydrylglnhibitors Iodoacetamide and p-chloromercuribenzoate (PCMB) were used at concentrations of 2.4 x 10-7M in enzyme solutions of pepsin, BP-13 protease and papain (Calbiochem) adjusted to pH levels of 2.5, 3.0 and 8.0 respectively. Five m1 of inhibitor (4.8 x 10"7 M) and 5.0 m1 of enzyme (60 pg/ml) were mixed and incubated at 30 C for 1 hr and assayed at the appropriate pH using 1% BSA as substrate. 57 Carboxyl Inhibitor Synthesis.--Diazoacetoglycine methylester was synthesized by the procedure of Riehm and Scheraga (1965). A solution of 9.1 g of glycylglycine methylester hydro- chloride and 5 g of sodium nitrate in 40 m1 of 2 M sodium acetate was cooled in an ice bath. Two ml of glacial acetic acid was added and the reaction mixture allowed to stand for 2.5 hr at 0 C. The diazoacetoglycine methylester was extracted with 25 ml aliquots of chloroform. Petroleum ether (30 - 60 C) was added to the combined chloroform extracts until the solution became turbid. The mixture was allowed to stand overnight at room temperature and the resulting precipitate was filtered and dried over phos- phorous pentoxide for 12 hr. This material was then recrystallized from chloroform by the addition of petroleum ether and dried as before. Reaction With Enzymes.--The BP-l3 protease, trypsin and pepsin were dialyzed against distilled water for 72 hr to remove inorganic ions such as chloride and sulfate which can promote decomposition of diazoacetoglycine methylester, according to Means and Feeney (1971b). Perchloric acid was then used to adjust all enzyme sols (100 pg/ml) to pH 5.0. Two mg of inhibitor was added to 5 m1 of each enzyme sol and incubated for 12 hr at 30 C. Pepsin, BP-13 protease and trypsin were then assayed for activity at pH levels 58 of 2.5, 3.0 and 8.0 respectively using 1% BSA in Universal buffer. Effect of EDTA and Calcium $013 of the BP—13 protease were made 10 mM with respect to disodium EDTA and calcium chloride, then assayed for activity using 1% BSA in Universal buffer at pH 3.0. Milk Clotting and Proteolytic Activigy of BP-13 Protease Milk Clotting The assay for milk clotting activity was based on the time in sec required to form curd fragments when 1 m1 of enzyme sol was added to 10 m1 of a 5% sol of skimmilk powder. The low temperature non-fat dry milk was dispersed in 0.03 M acetate buffer at pH 5.5 (p = 0.3) containing 10 mM calcium chloride. Milk clotting activity (MCA) was defined as where "t" is the time in sec required for curd formation (Kawai and Mukai, 1970). TCA Soluble Nitrogen As soon as the milk had clotted, 2.5 m1 of the coagulated solution was added to 3.0 m1 of 6% TCA. The soluble nitrogen for both calf rennet and the BP-l3 protease were determined by the Lowry-Folin method. 59 Proteolytic activity of the two milk coagulants was followed over a period of 1 hr by incubating an appropriate aliquot of enzyme with a 1% sol of casein at pH 5.75 and sampling at intervals of l, 5, 10, 20, 40 and 60 min. Enzyme concentrations were adjusted so initial rates of hydrolysis were approximately the same after 1 min. TCA soluble nitrogen was determined as above. Gel Electrophoresis of Casein Hydrolysates Approximately 100 units of enzyme was added to 10 ml of a 5% casein solution which was incubated at 30 C. One- half ml samples were taken at l min, 5 min, 10 min, 30 min, 3 hr and 20 hr and added to 0.5 m1 of spacer gel buffer containing 7 M urea. Thirty—five pl samples was applied to the slots with the exception of the control in which case 15 pl of a 5% casein sol were added. Both the 5% spacer gel and 8% running gel contained 6M urea. Alkaline gel electrophoresis was then performed as previously described. Evaluation of Milk Clotting Ability The BP-13 protease was evaluated as a calf rennet substitute in small scale cheese making experiments. Twenty 1b batches of whole milk (pasteurized at 145 for 30 min) were added to stainless steel containers and incubated at 30 C. The milk was inoculated with 1% starter culture (Streptococcus lactis) and after an increase of 0.02% in titratable acidity, the milk was divided into 60 two 10 1b batches. At this point, 0.9 ml of single strength calf rennet was diluted with 50 parts of water and added to one lot of milk. The amount of BP-13 protease added to the remaining batch of milk was equated to calf rennet on the basis of TCA soluble nitrogen produced in nine min using the standard assay procedure previously described. Five ml of CFE concentrate, obtained by ultrafiltration, corresponded to approximately 0.5 m1 of single strength rennet in terms of proteolytic activity. RESULTS AND DISCUSSION Enzyme Production Detection of Proteolysis When proteolysis of casein occurs in agar medium, a white zone of precipitation is formed due to the deposition of insoluble para-caseins mainly "para—K-casein." The extent of proteolysis exhibited by the Penicillium roqueforti is reflected by both the size and type of precipitation (Martley e£_g£., 1970). The four types of media are compared with respect to clarity of zone of precipitation and amount of mold growth (Table 1). Types III and IV media supported the best growth of all strains of P. roqueforti. This is to be expected as both media are formulated specifically for fungal growth. The Martley media (Types I and II) are used mainly for the detection of proteolytic organisms in total bacterial counts. Type I medium was autoclaved and this resulted in some visible precipitation of the casein. As a conse- quence, the zone of precipitation was masked to a certain extent and was not as sharp as in the case of Type II and III media where the casein was sterilized by a combination of hydrogen peroxide and low heat treatment. 61 62 TABLE l.--Qualitative detection of proteolysis by P. roqueforti (BP- 13) when grown on various -types of media. Growth of E. Clarity of Zone Medium roqueforti (BP-l3) of Precipitation I. Martley ++a Sharp (Autoclaved) II. Martley ++ Very Sharp (H202) III. Czapek-Dox +++b Very Sharp IV. Meyers +++ Medium White: Zone (H O ) of Precipitation 2 2 Unclear a++ Very good. b+++ Excellent. Type IV medium was unacceptable for detecting proteolysis due to opaqueness which formed as soon as the calcium chloride was added to the liquid medium. Trace quantities of metals such as Zn, Cu, Mn and Mo in the medium may have contributed to the partial precipitation of casein at this point. When 3. roqpeforti was streaked on this medium, the zone of precipitation was unclear. Type III (Czapek-Dox) was selected as the best medium for detecting proteolysis. All three strains of E. roqueforti (ATCC 10110, ATCC 6987 and Japan BP-13) produced a zone of precipitation on the agar medium. This zone extended from 0.5 to 1.0 cm beyond the mycelial growth, 63 indicating that all strains were producing an extracellular protease or proteases. The zone of precipitation was approximately the same size with each of the three strains; however, the BP-13 strain exhibited slightly more proteoly- sis within the immediate vicinity of the mycelium. This was evident from the clearing of the precipitated casein. The BP-l3 strain was selected for shake culturing experiments on the basis of the above results and earlier reports by Niki _£‘_l. (1966) in which the authors indi- cated this strain produced large amounts of extracellular protease. Choice of Broth for Shake Culturipg The BP-13 protease was grown on Czapek-Dox and Meyers broth at pH increments of 4, 5 and 6. Although the medium devised by Meyers and Knight (1958) was specifically formulated for the growth of P. roqueforti, no major differences were evident when compared to Czapek-Dox broth (Table 2). Both types of broth supported the best growth of fungi at pH 4.0. The Czapek-Dox broth was selected for culturing experiments on the basis that it was easier to prepare and could also be purchased commercially. Shake-Culturing_Experiments The fungi was cultured using varying amounts of added Tryptone and Proteose-Peptone No. 3 (Figure l and Figure 2, respectively). Analysis of the CFE for activity revealed that the broth containing 2.0% added Tryptone had 64 TABLE 2.--Effect of media and pH on the growth of P. roqueforti (BP-l3). Growth of g. roqueforti (BP-l3) Broth pH 4 pH 5 pH 6 Czapek-Dox +++b ++a ++ Meyers +++ ++ ++ a ++ Very good. b+++ Excellent. approximately three units/m1 after 86 hr of culturing while 0.5 and 1.0% Protease—Peptone induced the release of over 4.5 units of protease activity per ml during the same time period. When inducer was omitted from the Czapek-Dox broth, little proteolytic activity could be detected in the CFE. In succeeding culturing studies, 0.5% Proteose- Peptone was added to the liquid broth for two reasons: a. Isolation of an extracellular protease would entail removal of added inducer. b. This concentration produced 97% as much protease, after 86 hr of culturing, as did 1% inducer. The data in Figure 3, compares the various strains of Blue cheese mold with respect to their ability to ;produce extracellular protease. Both ATCC cultures failed 65 0—0 Control r—a O.I% Tryptone b—A 0.5 % Tryptone ActMIy O-——o I.O°/o Tryptone W” °——° 2.0% Tryptone 30 1 20 - IO - o n 1 72 96 I20 I44 I68 Time (hr) Figure 1. Effect of Tryptone on production of extracellular protease by P. roqueforti (BF-13) when shake cultured in Czapek-Dox broth. 66 AMWNy (unite/ml) °----* Control li———I O.I % Protease PepIane No.3 /" 4———¢ 05 °/o Protease Peptone No.3 °—————o I.O% Protease Peptone No.3 6.0 r- o———0 2.0% Ptoteoee Peptone No.3 .1 ' 4O - 2.0 r- / f ‘f A 0 so I00 I20 I40 :60 Time (hr) Figure 2. Effect of Proteose-Peptone no. 3 on production of extracellular protease by 3° Egggeforti (BF-13) when shake cultured in Czapek-Dox broth. 67 Activity (units/ml) —— Control 6.0 . ~——t Japan ,BP-l3 -——- ATCC .6987 °——-° ATCC , IOIIO 4.0 e 2.0 - O r- O 24 72 I20 l68 TIme (hr) FiSNJre 3. Production of extracellular protease by various strains of P. roqueforti during shake culturing in Czapek-Dox broth containing 0.5% Proteose- Peptone no. 3. 68 to produce significant quantities of protease while the Japanese strain showed high activity in the CFE. The BP-l3 strain of 3. roqueforti began to sporulate after 60 to 66 hr of shake culturing at 225 rpm and 25 to 27 C (Figure 4). Culturing was terminated after 72 to 78 hr. If culturing was extended beyond 80 hr, mycelial breakdown was extensive and preparation of the CFE was extremely slow due to clogging of the millipore filters. Prolonged culturing resulted in the release of excessive amounts of nucleic acids which tended to plug the G50 Sephadex column. This resulted in poor resolution of the three fractions. Problems with preparing a CFE were also encountered if culturing was terminated before sporulation had commenced. In addition, the yield of extracellular protease was also reduced (Figure 4). Infrequent transferring of the culture (once/3 months) resulted in a reduced amount of proteolytic activity in the CFE. Transferring weekly on Czapek-Dox agar con- taining 0.75% casein and 0.75% Proteose-Peptone no. 3, greatly increased the proteolytic activity but also created problems with respect to filtering and excessive amounts of nucleic acid. Sporulation was also delayed by 48 to 96 hr when the cultures were transferred on a weekly basis. A time interval of 3 to 4 weeks between each transfer was found to be ideal for maintaining proteolytic activity and minimizing the problems of filtering and excessive nucleic Activity (units/ml) 6.0 '- 4.0‘ 2.0 - Figure 4. 69 ‘ Beginning of Sporulation g Terminatlon of Culturing ' v ' v \ 9202020202.}. ‘ v A A ‘ //////////////////////I. :z: 4:223:29 V ‘ e'e'e Jv’ v v v V $202020: 9.. vvv .. A. A ‘0 vv 9. ‘. ’e .20. ‘ v Q 48 Time (hr) 1 96 UnIIs of Enzyme pH 0 I44 Production of extracellular protease and change in pH by E. roqueforti (BP-13) during shake culturing in Czape -Dox broth containing 0.5% Proteose-Peptone no. 3. 70 acid production. Protamine sulfate partially removed the nucleic acids but also inactivated the protease. A spore count of 90 to 100 x 106/300 ml of broth produced the desired amount of growth. Inoculations using a lower count resulted in prolonged culturing and the pro- duction of excessive amounts of nucleic acids. This latter problem appeared to be a consequence of mycelial lysis. Normally 15 one 1 flasks, each containing 300 m1 of broth, were shake cultured at a time. The yield of CFE varied from 3.5 to 4 2. The pH of the CFE was in the range of 3.2 to 3.5 (Figure 4). Determination of Assey_Parameters Effect of Substrate Concentration andng on Protease ActiVity The rate of hydrolysis of 0.5% casein at pH 4.0 was too slow to be considered a useful assay procedure for pro- teolytic activity (Figure 5). Using the same protein con- centration at pH 5.75 resulted in the release of consider- ably more TCA soluble nitrogen. This may be attributed to approaching the pH optimum for the enzyme more closely or could possibly be a function of the amount of protein in suspension. At pH 4.0, a major portion of the casein had precipitated out of solution. When the enzyme was assayed at pH 5.75, in the presence of 1.0% Hammersten casein, even larger amounts of TCA soluble nitrogen were released. This curve was more linear than when 0.5% casein was assayed at the same pH. Absorbance (600nm) 0.50 - 0.25 - 0 Figure 5. 71 0.5% casein , pH 4.00 0.5% casein , pH 5.75 »———-x |.0°/o casein , pH 5.75 I l I IO 20 30 Time (min) Increase in TCA soluble nitrogen (Lowry-Folin) resulting from hydrolysis of casein by the BP-13 protease (CFE) at 30 C. 72 A decision was made to use 1.0% casein at pH 5.75, on the basis of the results in Figure 5. The reaction was terminated after 9 min at 30C. Allowing hydrolysis of casein by the BP-13 protease to extend beyond 9 min resulted in pronounced non—linearity of the curve. Assay times less than 9 min resulted in less product (TCA soluble nitrogen) being formed and a corresponding increase in experimental error. The enzyme solution being assayed was diluted to the extent where a AA of 0.25 to 0.30 would not be exceeded in 9 min (100 to 120 pg of TCA soluble nitrogen). This was to insure that zero order kinetics would be approached as closely as possible throughout the entire assay procedure. When working with purified enzyme preparations, a AA of 0.25 in 9 min corresponded to approximately 8 pg of enzyme; however, this was solely dependent on the specific activity of the enzyme being assayed. Optimumng and Stabilityytong The optimum pH, when using 1% BSA as substrate, was 3.0 (Figure 6). With casein, a pH of 5.5 appeared to be optimum (Figure 7). This same optimum was reported earlier by Niki E£.3£° (1966). At pH 5.25 the casein sol became turbid, with considerable precipitation occurring at pH 5.0. This results in occlusion of a large number of cleavage sites. Consequently, less TCA soluble nitrogen is released per unit of time even though the pH optimum may lie in this range. Obtaining 100% relative activity at pH 73 Relative Acflvfly CHO lOOI- 75r- 25h pH Figure 6. Activity of BP-13 protease (CFE) at various pH levels using 1% BSA as substrate. 74 Relative Activity (96) I00 t Casein Solution -—\ Turbid 75- Precipitation of casein 50 P 25 - O 1 J J 40 50 ea 10 pH Figure 7. Activity of BP-13 protease (CFE) at various pH levels using 1% casein as substrate. 75 5.5 may be a true optimum or a reflection of the amount of casein in suspension. The BP-13 protease was rapidly inactivated below pH 3.0 and above 6.0 when stored for 48 hr at 4 C (Figure 8). When the curves for optimum pH are compared with stability to pH, a marked similarity is evident: the portions of the BSA curve (Figure 6) between pH 2 and 3 and the casein curve between 5.5 and 7.0 (Figure 7) correspond closely to the curve for stability to pH in Figure 8. This indicates that activity below pH 3.0 and above 6.0 is reduced due to denaturation of the enzyme. A decrease in activity between 3.0 and 5.0 when using BSA, may be due to a true reversible affect of the velocity, or an affect of pH on the affinity of enzyme for substrate. This same reasoning may also apply to the pH optimum curve for casein (Figure 7) but an additional factor, the precipitation of substrate, must also be taken into con— sideration. Optimum Temperature and StabiIity to Temperature The relative activity of the BP—l3 protease at various temperatures appears in Figure 9. The enzyme has maximum activity at 45 C when using the standard 9 min end point assay. Above 45 C the enzyme is rapidly inactivated. This is more clearly shown by Figure 10 in which the rate of hydrolysis was followed at time intervals of 3, 6 and 9 min at each temperature assayed. The rate of hydrolysis 76 Relative Actlvity M) [00>- 75'- 50- 25 ”\I Figure 8. Stability of BP-13 protease (CFE) to pH. 77 Relative .NMWNy OH lOO 75 25 o 1 l 4 l 20 30 40 50 60 Temperature C Figure 9. Relative activity of the BP-13 protease (CFE) at various temperatures. 78 Absorbance (600nm) 0.4 - 46c 48 C 0.3 - 35 C 50 C 0.2 - 20 C O.| "' f fi 55 c O 3 6 9 Time (min) Figure 10. Rate of hydrolysis of casein at various temper- atures by the BP-13 protease (CFE). 79 proved to be linear up to 46 C but at 48 C some in- activation had occurred.1 At 55 C the enzyme is rapidly inactivated. Although the true initial velocity increases steadily as the temperature is raised, the amount of sub- strate transformed at any finite time first rises and then falls, giving an apparent optimum temperature. This optimum temperature is not constant but decreases as the time interval increases: at 55 C an assay time of 30 sec or less would be required, but at 46 C the assay time can be lengthened to 9 min (Figure 10). Dixon and Webb (1964a) indicate the effect of temperature on the velocity of enzyme reactions may be due to several different causes including, stability of the enzyme, enzyme-substrate affinity, velocity of breakdown of the enzyme-substrate complex, alteration of pK values, pH functions of any or all of the components and affinity of the enzyme for activators or inhibitors. Temperatures up to and including 45 C for 30 min had no effect on the activity of the BP-13 protease (Figure 11). Beyond 45 C the enzyme was rapidly inactivated. A plot of loge of residual activity after 3, 6 and 9 min of incubation at temperatures of 50.1, 53.2, 56.0 and 59.9 C, indicates that the denaturation is of the first order and irreversible (Figure 12), when exposed to heat at pH 5.0 1Hydrolysis was also linear with time at temperatures of 25, 30 and 40 C but these curves were eliminated from Figure 10 for the sake of clarity. Residual Activlty (96) IOO 75 50 r 25'- Figure 11. 80 j l L l 1 l 1 30.0 40.0 50.0 60.0 Temperature 0 Stability of purified BP-13 protease to temper- ature (Enzyme incubated 30 min for temperatures up to 45 C; above 45 C, samples were incubated 9 min). 81 L09. oIt) Refldual Activity 4.6 45.0 C or less ' ’ 50J C ‘ ' 53.2 c 3.8 - , 56130 31) - 599 C i— 252 30 6f) 90 Thne(nfln) Figure 12. Rate of thermal inactivation of BP-l3 protease at various temperatures. 82 in 0.04 M citrate buffer (p = 0.3). Because inactivation parameters vary greatly with pH, Dixon and Webb (1964b) indicate that caution should be exercised in drawing general conclusions from observations at only one pH. Other variables such as ionic strength, protein concentration and inhibitors also have to be considered. The amount of enzyme denaturated at any given time (T) for a specific temperature, can be calculated from the slope of the lines in Figure 12. A T (50% destruction 0.5 of enzyme activity) of 41.3, 18.7, 7.35 and 3.57 min for temperatures of 50.1, 53.2, 56.0 and 59.9 respectively, were calculated from the relationship where K is the rate of inactivation. There were insufficient data in Figure 12 to calculate a meaningful energy of denaturation (Ea) for the BP-l3 protease. Activation Energy for Hydrolysis of Casein The rate of hydrolysis (k) of casein increases in an exponential manner with the temperature according to the Arrhenius equation. k = A e-Ea/RT 83 A is a pre-exponential factor, Ea is the activation energy, k a gas constant and T the temperature in degrees Kelvin. When the log of the above equation is taken log k - -Ea l e " mirl+1°9eA and loge k vs % is plotted, the Ea can be easily calculated. The loge Of the initial velocities at temperatures of 20, 30, 35, 40, 46 and 55 C (Figure 10) were plotted against %. An Ea of 8000 cal/mole, for the hydrolysis of casein by the BP-l3 protease, was calculated from the slope of the curve presented in Figure 13. Purification of BP-13 Protease Concentration Several precipitating reagents were used in an attempt to remove the extracellular protease from the CFE (Table 3). Despite the fact that ethanol, methanol and acetone precipitated significant quantities of protein, a major portion of activity in both the precipitate and supernatant was destroyed. This can be attributed to denaturation of the protease by these solvents. Salt fractionation with ammonium sulfate (90% saturation) yielded a recovery of 56.9% of the initial activity. This compares very closely to a recovery of 56.5% when the CFE was concentrated by ultrafiltration and pervaporation (Table 5). When the same precipitated enzyme 84 Log Initial Velocity -2.0 '- -2.5 - Ea 88000 cal/mole -3.0 - -3 5 ~ 3.0 3.I 3.2 3.3 3.4 3.5 -l— (K")xl03 Figure 13. Arrhenius plot for purified BP-l3 protease using 1% casein (pH 5.75) a substrate. 85 TABLE 3.--Recovery of BP-l3 protease activity after precipitation with various reagents. % Recoverya Precipitating Reagent % V/V Precipitate Supernatant Ethanol 60 n.d.b 20.9 ' 70 18.5 n.d. 80 31.8 n.d. 90 19.8 n.d. Methanol 60 n.d. 1.8 70 n.d. 1.8 80 n.d. n.d. 90 n.d. n.d. Acetone 60 n.d. 1.2 70 22.7 n.d. 80 23.1 n.d. 90 24.2 n.d. Ammonium % Sulfate Saturation 40 1.7 60.6 50 2.3 56.4 60 3.5 49.0 70 13.1 39.4 75 38.7 23.9 80 45.0 2.4 85 53.9 2.5 90 56.9 2.0 aFrom 50 ml of CFE. bNot detectable. 86 and retentate concentrate were fractionated on G50/G100 Sephadex, the specific activity of the latter was much higher than with the ammonium sulfate cut. In view of these results, ultrafiltration and pervaporation were selected as a means of concentrating the enzyme preparation. When using the UM 10 membrane (1.5 x 10-3 pm pore diameter) in the Amicon ultrafiltration unit, from 20 to 30% of the original activity could be detected in the ultrafiltrate. When the PM 10 membrane (1.8 x 10-3 pm pore diameter) was employed, up to 80% of the original activity was present in the ultrafiltrate. These results would indicate that the BP-13 protease had a molecular weight of less than 10,000; however, experiments in gel chromatography and SDS gel electrophoresis indicated a molecular weight of 49,000 and 45,000, respectively. For a molecule of this size to pass through a membrane with a molecular weight cut-off of 10,000, may indicate: a. High axial ratio for the enzyme b. Enzyme is composed of sub-units 0. Reduction in molecular size due to shearing stress in ultrafiltration. No evidence was found to support either "a" or "b." There was about 20% loss in activity due to shear denaturation of the molecule. According to Charm and Lai (1971) this is to be expected. For a molecule to be reduced from a molecular weight in the range of 45,000-49,000 to less than 10,000 87 and still retain activity seems only remotely possible. No attempt was made to determine the molecular weight of the enzyme present in the ultrafiltrate. Pepsin has a molecular weight of approximately 35,000 and is retained to the extent of at least 95% on the PM 10 membrane while the retention on the UM 10 membrane is greater than 99%. After the CFE was concentrated from 1 1 to approxi— mately 50 m1, further concentration was achieved by placing the retenate in dialysis tubing and pervaporating to a volume of 10 to 15 m1. This final concentration step required 6 to 8 hr. At this point the sample was deaerated and applied to the top of the 650 Sephadex column. Con- centrated samples, exhibiting high viscosity, were dialyzed for 12 hr at 0 C and pervaporated back to 15 m1 prior to application to the GSO Sephadex. If a highly viscous sample was applied to the column, the eluate would drain away from the top of the G50 gel before all of the enzyme concentrate was delivered. This was attributed to the lack of an air tight seal between the sample applicator and the inside wall of the column. The viscosity of the concentrate appeared to be a function of the amount of sucrose not utilized by the fungus during shake culturing. Purification of the BP-l3 Protease by Gel Filtration A picture of the columns (2.5 x 45 cm and 1.6 x 100 cm), monitor, fraction collector and additional accessories used in purifying the BP-l3 protease appears in 88 Figure 14. A schematic of the same apparatus appears in Figure 15, showing the distribution of the three major peaks during the fractionation procedure. By the time the Proteose-Peptone fraction had reached the bottom of the CBC Sephadex column, the nucleic acid-protein peak was coming off the bottom of the G100 Sephadex with the enzyme being distributed between these two major peaks. At this point the LV-4 valve was rotated 90° so the Proteose-Peptone fraction would bypass the G100 column. A second eluate flask containing the same 0.04 M citrate buffer at pH 5.0 (p = 0.03) was connected to the LV-4 valve and automatically fed the G100 Sephadex column when this valve was given a quarter turn. The results in Figure 16 are a plot of A254, enzymatic activity and protein concentration (Lowry-Folin) for each fraction collected. The first peak displays high absorbance at 254 nm in relation to the amount of protein present, indicating the presence of nucleic acids. The second peak represents the enzyme fraction. There is an excellent correlation between A254, protein and activity for this peak. This suggests that a high degree of purification has been achieved at this point in the fractionation procedure. The third peak contained the Proteose-Peptone which was originally added to the Czapek-Dox broth. The size of this peak depended on the amount of Proteose- Peptone allowed to reach the 6100 column. Figure 14. Gel filtration equipment used to purify the BP-l3 protease: monitor, fraction collector, recorder, columns and ancillary equipment. 90 .mmmououm maimm mcflwwapsm pom poms ucoEmHsvm coaumuuaflw Ham «0 owumEanom .ma musmflm 382.00 5.83... 73:82 >3 «26> 05.2.63. 30:. 5205:204 0.22.2 . 3020.1 mTam .— occfimdbaoozta o o>_o> ¢I>._ o>_o> 05.2.33. 30:. 145° m a see. m w. 0000 00 O. .... no. 1.59.3.3 5.5.8 II. 1.59.8.2 cease an .3238 one 38.38 00.0 fl fiI—i c.0232... uh“ , _ Esau gateway. Eon. u 91 mg protein I- ..2 Absorbance 254nm Units/Tube Tube *——-* A254nm ~2ao 2.0 4, I0 I——--: Unlte of acIIvIIy ~——.mg protein (Lowry-Folin) LG 'I- 0.8 - I50 l.2 1'- 0.6 '- iOO 0.8 “I" 0.4 ~ 50 OAq-QZ T‘ OOJLOOL~ - 1 . 4. o 20 30 4O 50 60 Tube Na. (5ml ltubel Figure 16. Purification of BP-l3 protease by gel filtration with 650 and G100 Sephadex. 92 Further purification of peak 2 was achieved by applying this fraction to a second G100 Sephadex column (1.6 x 100 cm). The results in Figure 17 shows the presence of two well resolved peaks, with the first fraction being the enzyme. This purification step served to almost double the specific activity of the protease (Table 4). The second elution peak obtained from the G100 column (Figure 17) contained a minor fraction that had no proteolytic activity. The steps required in the purifying of the BP-13 protease appear in Figure 18. A purification summary for the BP-l3 protease appears in Table 4. The CFE was arbitrarily assigned a purification fold of 1.0. A major portion of the Proteose- Peptone fraction was removed from the CFE by ultrafiltration. This resulted in a 2.69-fold purification of the concen- trated retentate. The G50/G100 Sephadex chromatography proved to be a powerful purification step: the specific activity increased from 8.85 to 285, reflecting in an 87.0-fold purification. Again this was due mainly to the removal of the Proteose-Peptone. The final G100 column served to increase the specific activity to approximately 465 which represents a 14l-fold purification from the initial CFE. The 280/260 ratio of the final enzyme preparation was 2.01. Normally a pure protein has a 280/260 ratio of 1.75 but a higher value of 2.01 may be a reflection of a 93 .xmemnaum cone nuns QN 0.0 0.x. 0.0. 1%: 33:22: coflumuuafim Ham an ammououm malmm mo GOHpMOHMHusm 12.2252 62 3:. .RH oasmnm em o“ m. N. o u q q 1 . q q q q o 0 lil o o Nd..?o .> I cd..oo moiufl. mo.uQ_ Agitation. 529:. an. ill. 0.. 33:2. .0 5.5 Ti... Ec¢n~< oil. on: Ecemm 353334 £208 on. 94 Ho.m flea omv om.v Away oma m.v~ ommm o.Hm o.mm xocmnmom ooao . . o.sm mmm em.ae Logo mom 0.8m ommm e.em 0.04 xueeaawm ooa0\omu . . mo.m mm.m o.ooo o.oe m.om oamm vmm o.mH cowuauomm>uam can coaumuuaammuuas . . oo.H mm.m oom.m om.m ooa oovm v.m oooa uomuuxm comm HHOO COADmOHm muw>auom me as man >ua>oomm muflco HE pom oom nausm oamwoomm Hmuoe oz ucoopom Hmuoe mafia: AHEV omm paom mESHo> moum cowumowmausm aflououm mua>fiu04 .omeauoum maimm mo ahaEESm cowuaowmwusmnthNcmll.v Manda 95 CULTURE Czapek-Dox Broth, 0.5% Proteose-Peptone, 90 to 100 x 106 spores/ 300 ml. Shake culture 72 to 76 hr at 25 to 27 C, 225 rpm. Centrifuge at 1000 G. ‘ v SUPERNATANT Vegetative growth (discard) Millipore filter, 0.8 pm Millipore filter, 0.45 pm V \V CELL FREE EXTRACT (CFE) Retentate (discard) Ultrafiltration, Amicon UM 10 membrane Concentrate 1000 ml CFE to 50 ml. \ 'W RETENTATE Ultrafiltrate (discard) Pervaporate 50 to 15 ml. \ TV CFE CONCENTRATE CFE Concentrate (low viscosity) (high viscosity) Dialyze 12 hr <_ Pervaporate to 15 ml Deaeration, 35 C G 50 and G 100 Sephadex columns in series (2.5 x 45.0 cm) V ‘W PEAK 2 (Enzyme) PEAK 1 (Nucleic acid - protein) Dialyze 24 hr PEAK 3 (Proteose-Peptone) Freeze dry G 100 Sephadex (1.6 x 100 cm) \ \V PEAK 1 (Enzyme) PEAK 2 (Small MW fraction) Dialyze, 36 hr Freeze dry ENZYME Figure 18. Steps in the purification of BP-13 protease. 96 higher tyrosine and/or tryptophan content. The 280/260 ratio was determined using a Beckman DU-Z spectrophoto- meter. Absorbance Spectrum of Nucleic Acid- Protein and Enzyme Peaks A scan of the first peak obtained off the GSO/GlOO columns showed the presence of two overlapping peaks with adsorbance maxima near 262 and 275 (Figure 19). This corresponds closely to the absorbance maximum of nucleic acid and protein respectively. A slight decrease in absorbance in the range of 266 to 270 nm indicates the area of overlap of the two spectrums. The BP-l3 protease had an absorption maximum at 280 nm. This is characteristic of most pure proteins. Standard Plate Counts Standard plate counts were made on several enzyme preparations after each operation in the purification procedure (Table 5). The CFE, UM 10 retentate and per- vaporated enzyme preparation had a count of less than 30/ml. The CFE is essentially sterile: the 0.45 pm Millipore filter removes all microorganisms with the exception of some viruses. From this point on, microbial counts are mainly a function of sanitation and pH of the enzyme solution. The very low microbial count for the UM 10 retentate and pervaporated enzyme preparation can be partially ascribed to the pH of the solution (3.2 to 3.5). 97 .AH xeuauuxmemaaum ooeoc ummuuopa maumm emamapsa cam AH xmomiixocmcmom ooa0\omov samuoumipaom camaosc mo Ednuoomm coflumHOmnm .ma mpsmam .65 £05.26; oon oom com com Com com com . a Z . a a a O .. mN_.O A. .32: noomd 5203.686 222.2 A _ .33 o 9822.. 9:3 3:30. 3.13.82 98 TABLE 5.--Standard plate count of BP-13 protease during purification procedure. Purification Step Count/ml BP-l3 (CFE) < 30 UM 10 Retentate < 30 Pervaporation < 30 G50/6100 Sephadex 50 - 200 G100 Sephadex 380 - 760 Low microbial counts in the fractions obtained from the various columns is to a large degree, a reflection of proper sanitation. Microbial counts for the final enzyme preparation ranged from 380 to 760 per ml. Electrophoresis of Various Fractions Resolved by Gel F1ltration The components of the first two peaks collected from the GSO/GlOO columns were subjected to alkaline acrylamide gel electrophoresis (Figure 20) using the system described by Melachouris (1969). The nucleic acid—protein peak (PK 1) was resolved into two very light bands (indi- cated by arrows in Figure 20) differing only slightly in electrophoretic mobility. Large quantities of sample (65 p1 of a 5% nucleic acid-protein solution) had to be introduced into the sample slot in order to achieve the straining effect presented in Figure 20. Figure 20. 99 Alkaline acrylamide gel electrophoresis of fractions collected from 650/6100 Sephadex. Nucleic acid-protein fraction (PK 1); first 3/4 of enzyme fraction (PK 2), tail of enzyme fraction (PK 2T) . 100 When the first 3/4 of the enzyme fraction (PK 2) was subjected to electrophoresis, a single band was obtained (Figure 20). The remaining 1/4 (based on volume) Of the enzyme fraction (PK 2T) produced two well resolved bands. The slower moving band possessed activity but the faster moving component was unable to hydrolyze casein. The final enzyme preparation was homogeneous when subjected to acid, alkaline and SDS gel electrophoresis (Figure 21). SDS gel electrophoresis was performed by adding 0.2% SDS to the enzyme sol prior to electrophoresis in an alkaline gel. When 0.1% mercaptoethanol was added to a 5% sol Of the BP-l3 protease and electrOphoresed in an alkaline gel, approximately 14 bands were resolved (Figure 22). This would suggest the presence of a large number of inter- chain disulfide bands. To have a proteolytic enzyme composed of 14 or more polypeptide chains covalently linked to each other by disulfide bonds, seems highly improbable. Determination of the number of polypeptide chains would require N terminal analysis. This experiment was not performed. The Molish test qualitatively indicated the presence of carbohydrate in the BP-l3 protease. According to Purkayasta e£‘_1. (1967), the carbohydrate prosthetic group of K-casein causes the formation of multiple bands when this milk protein is subjected to electrophoresis. 101 ALK ACID SDS Figure 21. Alkaline (ALK) , acid and SDS acrylamide gel electrophoresis of purified enzyme preparation. 102 E E-l-M Figure 22. The affect of 2-mercaptoethanol (M) on the BP-13 protease (E). 103 The carbohydrate of the BP-13 protease may also be producing a similar affect in this case. Further Purification of the BP-I3 Protease Alkaline, acid and SDS gel electrophoresis of the final enzyme preparation indicated the protein was homo- geneous. However, when the same protein sample was re- applied to the G100 Sephadex column (1.6 x 100 cm), two peaks were obtained. Both fractions had the same Kav as in the previous run but the specific activity of the enzyme had decreased from 477 to 343 (Table 6). In addition, the size of the second peak increased considerably while the quantity of enzyme decreased accordingly. The component(s) in the second peak did not possess proteolytic activity. When this fraction was added back to the enzyme, represented by Peak 1, there was no increase in activity. When the second peak (Figure 17) was subjected to either acid or alkaline gel electrophoresis, no stained areas could be located on the gel. Reversal of the electrodes and staining each gel type with either Amido Black or Coomassie Blue also failed in locating this component. This fraction had previously given a positive response to the Lowry- Folin procedure for measuring protein. These results indicated this non-staining species was a very small molecular weight component which was present as an impurity or formed due to autolysis of the enzyme. 104 TABLE 6.--Effect of repeated chromatography on specific activity of BP-13 protease. Column Size Step Gel Type (cm) Specific Activity 1 G50/6100 Sephadex 2.5 x 45 350 2 G100 Sephadex 1.6 x 100 477 3 G100 Sephadex 1.6 x 100 343 Autolysis was studied by incubating the BP-l3 protease at 30C for 30 hr. Protein, activity and TCA soluble nitrogen were determined at time intervals of 2, 18 and 30 hr. The results in Figure 23 show the protein level decreased approximately 9% after 30 hr but there was only a 4% drop in activity over the same time period. No reason can be given for this decrease in the protein level. In addition, the TCA soluble nitrogen continued to decrease throughout the incubation period. This negates the possi- bility of autolysis occurring during incubation in 0.04 M citrate buffer at pH 5.0 (p = 0.3). The amount of TCA soluble nitrogen should remain the same in the absence of autolysis. No plausible explanation can be given for this decrease. A Kav of 0.89, for the second peak obtained off the long G100 Sephadex column, indicated this fraction had a molecular weight of less than 1000 (Figure 26). When the BP-l3 protease was subjected to descending chromatography, five ninhydrin positive areas were detected (Figure 24). 105 Relative Change (70) IOOK) - 90!) ._I 80.0 °——° Activity °—° Protein 70,0 - *——-* TCA Soluble Nitrogen ‘7 0 IO 20 30 Time (hr) Figure 23. Change in characteristics of purified BP-13 protease after incubation in 0.04 M citrate buffer (pH 5.0, p = 0.3) for 30 hr at 30 C. 106 D 0 ‘— Origin 01> Om .0 Q @ ®® @o S a a 0 o 9 a a a 8 o o e Figure 24. Descending paper chromatogram of purified protease after various treatments: A, dialysis, B, precipitation with ammonium sulfate (85% satu- ration at 25 C); C, electrodrolysis at 225 V for 3 hr; and D, electrodialysis at 400 V for 18 hr. 107 The small molecular weight components would then appear to be either amino acids and/or small peptides. Electrodialysis and precipitation of the protease with ammonium sulfate (85% saturation at 25 C) failed to remove the amino acids and/or peptides (Figure 24). Electrodialysis at 225 V for 3 hr had no affect on the specific activity of the BP-l3 protease (Table 7) but did produce an additional spot on the descending chromatogram (Figure 24). A potential of 400 v for 18 hr reduced the specific activity over two-fold (Table 7) and also resulted in the formation of nine ninhydrin positive areas on the descending chromatogram (Figure 24). Electrodialysis at high voltage for a prolonged period of time results in precipitation of the enzyme from solution. This suggests the enzyme is a globulin. A decrease in activity of the BP-13 protease by electrodialysis can then probably be attributed to alteration of the secondary and teritary structure of the enzyme rather than disruption of covalent bonds. The equipment used for electrodialyzing the protease appears in Figure 25. An attempt was made to further purify the BP-l3 protease by using several types of ion exchangers (Table 8). Preliminary experiments were conducted to determine the optimum pH for adsorption of the enzyme to DEAE and SE cellulose. The results in Table 9 indicate that a pH of 7.0 was conducive to total binding of the enzyme to DEAE cellulose while 91.5% of the protease is adsorbed by the 108 TABLE 7.--Effect of electrodialysis on specific activity of BP-13 protease. Specific Time (hr) Voltage Activity Dialysis 24 . . 500 Electrodialysis 3 225 500 Electrodialysis 18 400 220 SE cellulose at pH 3.0. The isoionic point of the enzyme then lies somewhere between pH 3.0 and 7.0. Approximately 97% of the enzyme was adsorbed to the DEAE cellulose at pH 5.0 and 6.0. This indicates that a major portion of the enzyme was still negatively charged at each of these pH levels. Consequently, the isoionic point for the enzyme can be restricted to a pH range of 3.0 to 5.0 rather than 3.0 to 7.0. Binding experiments were not conducted with the remaining three cation exchangers. Each type of ion exchanger was used under varying conditions of pH and/or ionic strength. The results tabulated in Table 10 depict the problems associated with trying to further purify the BP-13 protease by ion exchange chromatography. The enzyme was tenaciously bound to the ion exchanger and as a result little or no activity or protein was recovered regardless of the method of elution. The best results were obtained when the enzyme was placed on a SE cellulose column equilibrated with 0.02 M citrate buffer (pH 4.44, u = 0.02) and eluted with an ionic strength 109 Figure 25. Electrodialysis equipment used in removing peptides and amino acids from BP-l3 protease. 110 ummwsn m asaunaaaswm a m in o.av tolmuo-m omaumH Homz z m H8 oom Am m muflauonfid r cowumu ummmsn Edfiunwaflsqm a N An o.m loooim omiomH . Hum ZN HE oom Am ouwHHmQE¢ u cofiumu O m cm I : wWOHDHHQU nommsn paw iOrmnonm Amy pans Edwnnaaflsqm a m o.~uo.a m oauonmmonm I cowumu o ummmsn o.Hv iOiWIOINmUIOIm mmoasaamo Ammv asaunaaasem a m m Hanomouasm . coaumo Hommsn m m N m omoasHHou Esfiunfiaflaqo a m m.m A mmmuvmz mu: mUIOIm Ammmov Hanna + ocwfim Hunuofla i scans Mm msouu Hmcowuocsm somcmnoxm coH mo om>e .ommououm marmm mo cowumuwuflusm 0:» ca poms mummcmnoxm GOw mo GOwumumcmqmu mo oozuoe paw moflumwuouomumnolr.m mqmde 111 TABLE 9.--Residual activity in supernatant after mixing of BP-13 protease with DEAE and SE cellulose at various pH levels. Residual Activity in Supernatant (%) Ion Exchanger pH 3 pH 4 pH 5 pH 6 pH 7 DEAE Cellulose 29.1 11.6 2.9 2.9 n.d.a SE Cellulose 8.5 16.8 24.1 31.6 . . aNot detectable. gradient. Under these conditions, 18.2% activity and 26.5% protein were recovered. This was totally unacceptable as a purification step due to the high loss of protein and activity. All attempts to further purify the BP-l3 protease were abandoned at this point. Determination of Molecular Weight A molecular weight of 49,000 was obtained for the BP-13 protease, using gel filtration (Figure 26). When SDS acrylamide gel electrophoresis was used, a molecular weight of 45,000 was obtained. This value was based on the BP-l3 protease having the same electrOphoretic mobility as ovalbumin. Attempts to establish a standard curve using aldolase, ovalbumin, chymotrypsinogen A, pepsin and ribonuclease A were unsuccessful: aldolase would not enter the acrylamide gel; chymotrypsinogen was impure and ribonuclease could not be located on the stained gel regardless of how much protein was applied. 112 uemnemum No.0 u : m.o~ «.ma o.~ n : numemuum ev.v mm vacoH mumuuao Smo.o ucmflemum mmoo.o u a m.m m.e m.o u : numcmoum m.m mm oficoH mumuuflo moo.o nemaemum mao.o u : .o.c .e.a o.~ u 1 cumcmuum m.m mm oflcoH mumuuwo Emo.o omoHsHHmu mm .©.: .p.c o.m mm AU .©.c .p.: o.v mm Au .0.6 .v.c o.m mm an cowusam .c.c .e.c 0.0 mm A6 mm H. u a no as om omfl3mmum .o.c .e.e ~.m u 3 Am .oé 6.: eta u a 3 .6... do 6.6 u a a .p.c .p.c v.0 u 1 Au coausHo .e.e .e.c ~.o n a in numcmuum ammo.o u n .p.c .p.c H.o u n An owsoa m.m mm omoasaawo .o.c m.u.c m.m mm mo HE om omw3mmum mumuuwo zHo.o Imdmo cwououm mo >uw>fluod mo Howusm ocfiuflfiwq mo cofiusHm mommsm Homcmcoxm wum>oomm w >um>ooom a numcmuum UflcoH uo mm mo mama Edaunwafldwm coH mo mama .mnumcmuum owcow can mao>oa mm msoaum> um Hmwmsn mumuuflo nuw3 mcflusam c053 mummcmnoxo cow mmoasaawo Scum cwwuoum can hufl>wuom ommmuoum maimm mo >Hm>oommll.oa mamae 113 .waneuumuoe uoc .0.em w.o n n ucmflpmum m.¢m N.m N.m mm numamuum mumuuwo 2H.o owcoH\mm m.o u : ucmflpmum .0.c .0.c H.m mm nuocmuum oumuuao 200.0 oncoH\mm m.o u : .p.c .p.s H.m mm unmaomum mumuuwo Smo.o mm ucoapmum .©.c .©.: m.o n : nuvcmuum oacoH m~a0.0 u : 0.m me mumuuao 2H0.0 mma0.0 n : o.m mm mumuuao 2H0.0 000.0 u n 0.0 mm mpmuuflo 2000.0 0~H.mH euaaumeee 0mtomH moaaumnsa mmoasaamo m 114 Molecqu Weight (x :0”) 20 r Aldolaee MW. l58,000 to b BP-I3 Protease M.W.49,000 8 .- 6 l- 5 .- 4 L Ovalalbumln MW. 45,000 3 .— hymotrypelnogen A MW. 25,000 2 i- Rlbonucleaee M.w. l3.700 I l 1 l l l 1 0| 0.2 0.3 0.4 0.5 0.6 K av Figure 26. Standard curve for determining molecular weight by gel filtration using G-lOO Sephadex (1.6 x 100 cm column). Kav = Ve - Vo/Vt - Vo. 115 Inhibition Studies Serine Protease Inhibitor The electrophilic sulfur atom of phenylmethyl sulfonylfluoride reacts with the nucleophilic hydroxyl group of serine, eliminating a fluoride ion and forming a stable derivate of the enzyme: =0 II ¢-CH2- -F + Protein-OH -—————9 Protein-S-CH2-¢ + HF O O: This derivate is similar in some respects to the acyl intermediates formed in the hydrolytic reactions of serine proteases (Means and Feeney, 1970a). Phenylmethyl sulfonylfluoride appeared to have a stimulatory affect on both pepsin and the BP-l3 protease (Figure 27). Ryle (1970) indicates that no activators of pepsin have been reported to-date. Therefore, the apparent increase in activity is most likely due to modification of the substrate (1% BSA) rather than activation of either pepsin or the BP-13 protease. The phenylmethyl sulfonyl- fluoride made have denaturated the BSA and exposed additional cleavage sites. This would then result in larger amounts of TCA soluble nitrogen being released and produce an apparent increase in activity. Apparently the BSA should have been denatured by heating. 116 .sflmmmuu paw ammmuoum malmm .cflmmom mo mua>fluom any so mpwuosHMHMGOMHSm HwnuaEHmcosm mo Hummus .nm ansmflm 0.0%.... 0”.an 0.000“. * - O - 0N 8.3052230 262.22.... . 00 20:0. x 3.. 8.523850 ‘\ $502.20.... “ 1 ob .. x ._ \ .2 n o. o W .2200 f .l L L 00. as $52 050.0". 117 Trypsin, a serine protease, was almost totally inhibited (97%) when exposed to phenylmethyl sulfonyl- fluoride for 20 min at 25 C (Figure 27). These results then indicate that the BP-13 protease is not a serine protease. Sulfhydryl Inhibitors Iodoacetamide and p-chloromercuribenzoate combine with the free SH groups according to the equations given below: 0 0 II II z-C-NH2-——4'Protein-S-CHz-C-NH2+HI 0 II II b) Protein-SH + Cl-¢-C-Hg——-) Protein-S-¢-C-Hg + HCl a) Protein—SH + I-CH The derivatives formed have an inhibitory action on sulfhydryl proteases such as papain (Figure 28) but the activity of an acid protease like pepsin is reduced by only 20% with iodoacetamide and 25% by p-chloromercuribenzoate. The activity of the BP-l3 protease was reduced 14% by iodoacetamide and 28% by p-chloromercuribenzoate (Figure 28). The free sulfhydryl groups of the BP-13 protease do not appear to play an essential role in the active site of this protease even though the activity was reduced to some extent. Derivatizing the SH groups undoubtedly produces conformational changes in the enzyme. This may in turn reduce the hydrolytic activity of the protease. 118 .cammam 0:0 ammmuoum malmm .cflmmmm mo >ua>fluom 050 so oumoNcobflusoHoEouoHnolm cam moHEmumomooow Mo uoammd .mm musmflm 588 0.10 see... 0 L 00 200200.500... 0.0.50 2 #0. x 0.. l 00 0222000000. . 2 b.0330 “ 1 0» a .2200 L .l l .. Co. as 33:2 2:23. 119 Carboxyl Modification Means and Feeney (1970b) indicate that diazoacetates are very specific for carboxyl groups of proteins. The reaction takes place under mild conditions: O Protein-C-OH + :fi=fi=CH-C-NH-CH2-c-o-CH3 Aspartic or (Diazoacetoglycine methylester) glutamic carboxyl group Perchloric acid / NaOH pH 5.0 S? 9 S? Proteln - C-O-CHz-C-NH-CHz-C-O-CH3 + N5 Doscher and Wilcox (1961) found the unionized carboxyl group to be the reactive species. The optimum pH for reaction with carboxyl groups is near pH 5.0 according to Riehm and Scheraga (1965). At lower pH levels, diazo- acetates hydrolyze easily and limits the extent of modifi- cation (Means and Feeney, 1971b). Due to the highly reactive nature of diazoacetates in aqueous solution, excess reagent must be added. Perchloric acid and its salts do not react with diazoacetates and can be used to adjust the pH or ionic strength of the reaction solutions. The decomposition of diazoacetoglycine methylester can be followed easily: the solution will turn from yellow to colorless and nitrogen evolution will cease. The proteolytic activity fo both pepsin and the BP—13 protease was inhibited to the extent of 91.5 and 91.9% respectively by the diazoacetoglycine methylester 120 (Figure 29). These results strongly suggest the presence of at least one carboxylic amino acid residue at the active site of the BP-l3 protease. Trypsin was unaffected by the diazoacetoglycine methylester (Figure 29). Effect of EDTA and Calcium Enzyme sols, 10 mM with respect to EDTA and calcium chloride, exhibited the same proteolytic activity as the controls. The BP-13 protease then does not have a de- pendency on divalent cations for activity, nor does Ca stimulate protease activity. Hydrolysis of Synthetic Peptides and Amino Acids Esters by BP-13 Protease Many enzymes have Km values as low as 1 x 10"3 M. For this reason each substrate was used at a concentration of 10 x 10'3 M (10 mM) to insure that zero order kinetics would be attained in the event of hydrolysis. Normally substrate concentrations of 10 to 100 Km are employed in most assay procedures. Of the 15 synthetic substrates listed in Table 11, only L-leucyl-L-tyrosine was hydrolyzed. In this case, 1.9% hydrolysis occurred after incubation for 1 hr at 30 C. This was considered too slow to be of any value in an assay procedure. Failure of the BP-13 protease to hydrolyze any of the remaining 14 substrates may indicate: 121 .cflmmmuu paw ommmuoum maumm .cflmmmm mo mufl>fiuom any so noumoawzuwa mcflohamoumomonfio mo pommwd .mm musmflm 535 0.10 58.. w m B o .. mm - on .. on 00.00.2005. “ 006202000085 “ x . \ 2 mac. w. \. L L L . 00. .2200 3.. 3.284 0.50.0: 122 TABLE ll.--Hydrolysis of synthetic peptides and amino acid esters by BP-13 protease.' % Hydrolysis Substrate Assay pH 3 4 5 N-CBZf-alpha Glutamyl-L-Tyrosineb n.d.e n.d. n.d. N-CBZ-alpha-L-Glutamyl-L-Phenylalanineac n.d. n.d. n.d. N-CBZ-Glycyl-L-Tyrosineb n.d. n.d. n.d. N-CBZ-Glycyl-L-Glutamic acida n.d. n.d. n.d. N—CBZ-Glycyl-L-Serinea n.d. n.d. n.d. N-CBZ—Glycyl-L-Tryptophanbc n.d. n.d. n.d. N-CBZ-G1ycy1-L-Phenyla1aninea n.d. n.d. n.d. N-CBZ-Glycyl-L-Leucinea n.d. n.d. n.d. N-Acetyl-L-Phenylalanyl-L-DiiodiotyrosineaCd n.d. n.d. n.d. L-Leucyl-L-Tyrosineb n.d. n.d. 1.9% L-Leucyl-L-Phenylalanineb n.d. n.d. n.d. L-Methionyl-L-Pheny1a1anyl-Glycineb n.d. n.d. n.d. L-Methionyl-L-Phenylalanineb n.d. n.d. n.d. Para Tosyl-L-Agrinine Methyl Ester n.d. n.d. n.d. N-Benzoyl-L-Arginine Ethyl Ester n.d. n.d. n.d. aDissolved in 0.03M NaOH at 35 C. bDissolved in 0.01M NaOH at 35 C. cOnly slightly soluble at pH 3. dModerately soluble at pH 4. en.d. = not detectable. f CBZ = Carbobenzoxy. 123 a. The enzyme was not specific for any of the amino acid combinations present and/or the peptides were too short for the enzyme to bind and hydrolyze. b. The N terminal carbobenzoxy group present on seven of the peptides may have prevented binding of substrate to the enzyme. The methyl and ethyl esters of arginine are hydrolyzed by trypsin, which is not only a peptide peptidohydrolyase but also an esterase. The BP-l3 protease does not possess esterase activity when p-tosyl-L-arginine methyl ester and N-benzoyl-L-arginine ethyl ester are used as substrates. Hydrolysis of the Oxidized B Chain of Insulin Two dimensional separation of the insulin hydroly- sates (B chain) was achieved by high voltage electrophoresis and descending chromatography (Figure 30). A total of 14 ninhydrin positive areas were identified on the peptide map. Twelve of these spots could be clearly identified while two areas were less distinct but still discernible. Free proline was identified by the color reaction to ninhydrin. The release of this amino acid would require hydrolysis of the peptide bonds between Thr-Pro (residues 27 and 28) and Pro-Lys (residues 28-29). The presence of 14 hydrolystate products indicates cleavage of insulin at 13 sites. The peptide map in Figure 30 is representative of the proteolytic nature of 124 Electrophoresis , SOV/crn , pH 3.6 v Q Descending Chromatography 1% @Q @ 2 Figure 30. Separation of insulin (oxidized B chain) hydroly- sates using high voltage electrophoresis followed by descending chromatography. (Dotted areas indicate low color intensity.) 125 the BP-l3 protease only if all bonds that are susceptible to cleavage are hydrolyzed to the extent of 100%. Incom- plete hydrolysis would yield a peptide map which over emphasizes the true proteolytic nature of the enzyme. Cleavage of the oxidized B chain of insulin at 13 sites would seem to indicate that the BP-l3 protease is very proteolytic; however, rennin has the ability to cleave this same substrate in nine places while pepsin can hydrolyze 10 peptide bonds. Despite the fact that both rennin and pepsin cleave several places, only two bonds are hydrolyzed beyond 20% by rennin when incubated for 20 hr under optimum conditions (Rickert, 1970). In this same paper, Rickert also points out that pepsin hydrolyzes four bonds between 10 and 20% with the remaining bonds being cleaved less than 10%. Mgggg miehei only cleaves 6 linkages of the B chain of insulin but hydrolysis at five of these sites is in excess of 20%. The most susCeptible site of attack was between Try (16) and Leu (17). Sixty— three percent of the B chain was cleaved between these two amino acid residues (Rickert, 1970). In a related experi- ment, McCullough and Whitaker (1971) obtained 9 spots on a peptide map of M2225 pusillus when incubated for 20 hr with the B chain of insulin. Analysis showed cleavage at 8 peptide bonds. In this instance, the peptide map was almost a quantitative reflection of the number of cleavage sites. In the case of rennin and pepsin, each of the peptides has 126 to be sequenced to accurately determine which peptide bonds are hydrolyzed. The BP-13 protease is considerably more proteolytic than pepsin, calf rennet or any of the major commercial fungal rennets. Consequently, the peptide map obtained in Figure 30 should be an accurate reflection of the number of peptide bonds cleaved by the extracellar protease of P. roqueforti. Com arison of the Proteolytic Action of tEe BP-13 Protease and Calf Rennet Hydrolysis of a 1% casein solution (pH 5.75) by the BP—l3 protease and calf rennet are compared in Figure 31. After 1 min and 5 min both enzymes had released approxi- mately the same quantity of TCA soluble nitrogen but between 5 and 60 min the BP-l3 protease produced a AA of 0.685 while calf rennet reflected a AA of 0.063. The high initial velocity by the calf rennet is due to rapid hydrolysis of the methionyl-phenylalanine linkage of K-casein. After the cleavage of this bond, hydrolysis is slow and non-specific. Hydrolysis by the BP-l3 protease appears also to be non- specific but very rapid compared to calf rennet. The portion of the curve between 20 and 60 min for hydrolysis of casein by the BP-l3 protease (Figure 31) is nearly linear even though the reaction is proceeding at a rapid rate. Again this indicates generalized proteolysis rather than specificity for a particular peptide bond. 127 Absorbance (600 nm) L ~ 00 *——* Calf Rennet o—-———-o BP-l3 Protease (175 (150 (125 l l l 0 20 4O 60 Thne(nfln) Figure 31. Hydrolysis of 1% casein (pH 5.75) with calf rennet and BP-13 protease. 128 Figure 32 represents the gel patterns obtained when the BP-l3 protease is incubated with 5% casein at pH 5.75. Samples were taken at 1 min, 5 min, 10 min, 30 min, 3 hr and 20 hr. After only 1 min both the as- and B-casein had changed slightly with a number of additional light staining bands moving ahead of the aS-casein. The 8- casein appears to be hydrolyzed slightly more rapidly by the BP-13 protease but after 20 hr both the as- and 8- casein are no longer identifiable on the alkaline gel pattern. Kappa casein remains virtually unchanged through- out the entire hydrolysis procedure. One major peptide, with high electrophoretic mobility, appears to be prefer- entially released after 1 min of hydrolysis. This band has the highest electrophoretic mobility and continues to increase in size up to 3 hr but after 20 hr this component also disappears. The gel pattern in Figure 32 confirms the experi- mental results obtained in Figures 30 and 31 in which the BP-13 protease was characterized as being very proteolytic, yet non-specific in its action on protein. The BP-13 protease would not be an acceptable calf rennet substitute on the basis of the gel pattern obtained in Figure 32 because both the as- and B-casein are ex- tensively hydrolyzed while K—casein is virtually unaffected. An ideal substitute is one which acts preferentially on K- casein but has little affect on either as- or 8-casein. 129 04....Q! : fig”; C IM 5M lOM 30M 3H 20H C Figure 32. Alkaline acrylamide gel electrophoresis of casein (C) and BP-13 hydrolysates of casein at various time intervals (M-min; H-hr). 3:7 ' t .J“: 12*. 2):; 'f 0.! U.” , ..’ < A: «5“ .I‘ 130 Evaluation of Milk Clotting Ability When the BP-13 protease was used at a concentration comparable to veal rennet, coagulation was very slow and a consequence of acid formation rather than a result of hydrolytic action by the fungal protease. The clotting time could be shortened to 1 hr by adding 10 times as much of the enzyme but this was still unacceptable: a. Clotting time was still too long. b. The milk never formed a smooth firm gel but tended to coagulate. This was followed by syneresis and precipitation of the casein. The BP-13 protease had a milk clotting activity/ proteolytic activity ratio of 0.005 while the ratio for calf rennet was 0.073 (Table 12). The fungal rennet is than 0.073/0.005 or 14.6 times as proteolytic as the calf rennet for the same milk clotting activity. 131 .mcfiuuoao mo 08H» 00 ommmoaou cmmouuflc oHQDHOm «08 00 m2 n . com 0 HO UM COH D .n X lelllyl fl 0 0 .0 0.0 000m 0020 000.0 000 00.0 00000000 manmm 000.0 000 0.00 000000 0000 m00>0000 00000000000 .000.0 000. .002. m00>00o0 000000 \002 0000>0000 00000000000 000000000 0002 .mmmmuoum malmm can 000000 mama mo m00>0000 Ufluwaomuonm 000 >00>0000 mcauuoau waE mo cOmwummEooli.~H mqmde SUMMARY AND CONCLUSIONS g. roqueforti (strain BP-13) was shake cultured in Czapek-Dox broth containing 0.5% Proteose-Peptone No. 3. The extracellular protease was isolated from a concentrated CFE by fractionation on a series of Sephadex columns. A 140 fold purification was obtained in the final enzyme preparation. The protease isolated was thought to be truly extracellular for three reasons: a. Culturing conditions were adjusted to minimize mycelial breakdown. b. The vegetative growth was centrifuged out and never subjected to induced cellular disruption. c. The enzyme contains carbohydrate, which is charac- teristic of most extracellular proteins. The pH optimum was 3.0, 5.5 and 3.0 to 4.0 for BSA, casein and the oxidized B chain of insulin respectively. Based on the pH optimum for BSA and insulin, the enzyme would appear to be an acid protease. This was confirmed in an experiment where the BP-13 enzyme was reacted with an 132 133 acid protease inhibitor (Diazoacetoglycine methylester) and found to be almost totally inactivated. Either aspartic and/or glutamic acid would appear to be involved in the active site of this enzyme. Serine and sulfhydryl protease inhibitors did not significantly reduce the action of the BP-13 protease when the activity of this enzyme was compared to pepsin, Ethylenediaminetetraacetate, a chelating agent, had no affect on enzymatic activity. Therefore, the enzyme does not appear to have a dependency on divalent cations for activity. The enzyme exhibited maximum stability to pH in the range of 3 to 6. Inactivation on either side of this range was due to denaturation of the enzyme. An optimum temperature of 45 to 46 C was obtained when the enzyme was assayed with 1% casein at pH 5.75. Inactivation, due to heat denaturation, was evident at 48 C when using the standard 9 min end point assay. Increasing the temperature beyond 48 C resulted in first order irreversible thermal inactivation of the enzyme. An activation energy of 8000 cal/mole was calculated for the hydrolysis of casein under standard assay conditions where the only variable was temperature. There was some doubt as to whether total purity had been obtained in the final enzyme preparation. Small molecular weight components, identified as amino acids and/or peptides, were present in the final enzyme 134 preparation. Extensive dialysis, electrodialysis ion exchange and precipitation of the BP-13 protease with ammonium sulfate failed to remove this fraction. Whether the small molecular weight components were present as impurities (from the Proteose-Peptone added initially) or were formed as a result of autolysis, was not resolved. Amino acids and peptides will not bind dyes such as Amido Black or Coomossie Blue. This may account for obtaining one band when the final enzyme preparation was subjected to acid, alkaline or SDS gel electrophoresis. The addition of mercaptoethanol to the protease resulted in the for- mation of 14 bands on an alkaline acrylamide gel. Either the enzyme is composed of a number of polypeptide chains linked to each other by disulfide bonds, or the large number of bands in the gel pattern are an artifact caused by the carbohydrate moiety associated with the protease. Molecular weights of 49,000 and 45,000 were obtained using gel filtration and SDS gel electrophoresis respec- tively. The action of the BP-l3 protease on 15 synthetic peptides and amino acid esters was very limited. Only L- leucyl-L-tyrosine was hydrolyzed to a detectable extent. The hydrolysis of this substrate was too slow to be of any value as an assay method for proteolysis. Failure to hydrolyze any of the remaining 12 peptides may have been a function of chain length and/or a lack of specificity for the amino acid residues present. Also the N terminal 135 carbobenzoxy group, present on 7 of the 13 peptides, may have blocked hydrolysis if these substrates by the enzyme under investigation. In addition, the methyl and ethyl esters of arginine were not hydrolyzed by the BP-l3 protease. Incubation of the oxidized B chain of insulin with the BP-l3 protease resulted in the formation of 14 ninhydrin positive areas on the peptide map of the hydrolysate. These results indicated the protease was capable of hydrolyzing a large number of peptide bonds. The peptide map was thought to be an accurate reflection of the number of bonds hydrolyzed. The accuracy of this map is totally dependent on the degree of hydrolysis of each cleavage site. The initial rates of hydrolysis of a 1% solution of casein by calf rennet and the BP-13 protease were nearly equal but between the time period of 5 and 60 min, hydrolysis of casein by the fungal protease proceeded at approximately 11 times the velocity of veal rennet. After hydrolysis of the methionyl-phenylalanine linkage of K- casein, the rate of hydrolysis of substrate by calf rennet fell off rapidly while the velocity of the BP-13 protease reaction remained almost linear with time. The affect of the Penicillium protease on the major casein components was clearly shown by acrylamide gel electrophoresis of the casein hydrolysates. After 20 hr of enzymatic action, both the as- and B-casein were no 136 longer evident on the gel pattern, but K-casein remained virtually unchanged. These results alone indicated the BP-13 protease was far more proteolytic than pepsin, chymosin or any of the commercially acceptable fungal rennets. The BP-13 protease was found to have over 14 times the proteolytic activity of calf rennet for the same milk in“ clotting ability. Calf rennet formed a smooth firm gel when the milk clotted. When the Penicillium protease was substituted for calf rennet, the milk tended to coagulate 1“; into distinct particles. This was followed by syneresis and precipitation of the casein from suspension. These results are a consequence of the proteolytic nature of the enzyme: a. K-casein appeared to be resistant to hydrolysis by the BP-13 protease while as- and B-casein were rapidly hydrolyzed. b. The enzyme acted in a very non-specific manner on a large number of peptide bonds. c. The protease was unable to hydrolyze the methionyl-phenyalanine linkage of a synthetic peptide containing these two amino acids. However, this may be a function of peptide chain length rather than specificity. 137 The action of the BP-13 protease on insulin and casein indicate the enzyme is an endopeptidase. An Enzyme Commission number (E.C. no.) of 3.4.4.99 can be applied to the enzyme with the information gained in this study. The numbers from left to right represent class (Hydrolase), sub class (Peptide hydrolase) sub-sub class (Peptide peptidohydrolase) and serial number. A serial number of 99 was assigned to the BP-l3 protease because the nature of the reactions catalyzed have not been fully investigated to determine which peptide bonds are hydrolyzed. 5. 4m “JV; . 2:04.0- 2.2:? BIBLIOGRAPHY BIBLIOGRAPHY Abdel-Fattah, A. F., Mabrouk, S. S., and El-Hawwary, N. A. 1972 Production and some properties of rennin like milk-clotting enzyme from Penicillium citrinum. J. Gen. Microbiol. 70:151-155. Alais, C., and Novak, G. 1970 Purification and properties of the milk- coagulating enzyme derived from Endothia parasitica. XVIII Int. Dairy Congr. 1E:279. American Public Health Assoc. Inc. 1960 Standard Methods for the Examinatign of Dairy Products. 11th Ed. Washington, D.C. 148 pp. plus XII. Arima, K., Iwasaki, S., and Tamura, G. 1967 Milk clotting enzyme from microorganisms. Part I. Screening test and the identification of the potent fungus. Agr. Biol. Chem. 31:540-545. Arima, K., Yu, J., and Iwasaki, S. 1970 Milk clotting enzyme from Mucor usillus var. Lindt, pp. 446-460. In: Methods in Enzymology. Vol. 19. Perlmann, G. E., and Lorand, L. (ed.). Academic Press, New York. 1042 pp. plus xx. Association of Official Agricultural Chemists. 1960 Official Methods of Analysis. 9th Ed. Associ- ation of Official Agricultural Chemists, Washington, D.C. 832 pp. plus xx. Babbar, I. J., Srinivasan, R. A., Chakravorty, S. C., and 1965 Dudani, A. T. Microbial rennet substitutes —A review. Indian J. Dairy Sci. 18:89-95. 138 139 Babel, F. J. 1967 Rennin-pepsin mixtures in cheese manufacture. Dairy Ind. 32:901-904. Bailey, J. L. 1967 Column chromatography of proteins, pp. 278-304. In: Techniques in Protein Chemistry, 2nd Ed. Elsevier, New York. 406 pp. plus xiv. Balls, A. K., and Hoover, S. R. 1937 The milk-cloting action of papain. J. Biol. Chem. 121:737-745. Behnke, U. 1967 Studies on the action of rennets of different origin. I. Milk coagulation and casein degradation. Milchwissenschaft. 22:563-569. Berridge 1954 Rennin and the clotting of milk, pp, 423—443, In: Adygnces Enzymology. Vol. 15. Inter- science Publishers, New York. 547 pp. plux x. Birkkjaer, H. E., and Thomsen, D. S. 1970 Experiences with some rennet substitutes. XVIII. Int. Dairy Congr. lE:275. Cerbulis, J., Custer, J. H., and Zittle, C. A. 1960 Action of rennin and pepsin on B casein: insoluble and soluble products. J. Dairy Sci. 43:1725-1730. Chapman, Helen R., and Burnett, J. 1968 A rennet/pepsin mixture for cheddar cheese. Dairy Ind. 33:308-311. Charm, S. E., and Lai, C. J. 1971 Comparison of ultrafiltration systems for con- centration of biologicals. Biotech. Bioeng. 13:185-202. Chaudhari, R. V., and Richardson, G. H. 1971 Lamb gastric lipase and protease in cheese manufacture. J. Dairy Sci. 54:467-471. Clark, J. M., Jr. 1964 Experimental Biochemistry. lst Ed. W. H. Freeman and Co., San Fransico. 228 pp. plus ix. 140 Coch Frugoni, J. A. 1957 Tampone universale di Britton e Robinson a forza ionica constante. Gazz. Chim. Ital. 87:403-407. Dan, H., and Jespersen, N. J. T. 1970 Rennet-pepsin mixture for cheese making. XVIII. Int. Dairy Congr. 1E:292. Dewane, R. A. 1960 Rennet substitutes. A review of literature. Paul Lewis Lab. Inc., Dairy Division, Milwaukee, Wis. 11 pp. Dixon, M., and Webb, E. C. 1964a Effect of temperature, pp. 145-166. In: Enz mes. 2nd Ed. Academic Press, New York. 950 pp. plus xix. Dixon, M., and Webb, E. C. 1964b Effect of pH, pp. 116-144. In: Enzymes. 2nd Ed. Academic Press, New York. 950 pp. plus xix. Doscher, Marilynn S., and Wilcox, P. E. 1961 Chemical derivatives of a-chymotrypsinogen. IV. A comparison of the reactions of a-chymotryp- sinogen and of simple carboxylic acids with diazoacetamide. J. Biol. Chem. 236:1328-1337. Dox, A. W. 1910 The intracellular enzymes of Penicilluim and Aspergillus, with special reference to those of Penicillium camemberti. U.S. Dept. Agr. Bull. 120. 70 pp. Dulley, J. R., and Kitchen, B. J. 1972 Phosphopeptides and bitter peptides produced by rennet. Aust. J. Dairy Tech. 27(2):10-1l. Dunn, M. S. 1949 Casein., Biochem. Prep. 1:22—24. Dutta, S. P., and Grzybowski, A. K. 1961 pH and acid-base equilibria, pp. 19-44. In: Biochemists Handbook. Long, E. (ed.). E. and F. Spon Ltd., London. 1192 pp. plus xxii. 141 Dutta, S. M., Kuila, R. K., Srinivasan, R. A., Babbar, 1971 I. J., and Dudani, A. T. Milk clotting enzymes from spore forming bacterial isolates. Milchwissenschaft. 26: 683-685. Edelsten, D., and Jensen, J. S. 1970 Investigations of microbial rennet from Mucor miehei. XVIII. Int. Dairy Congr. 1E:280. Edwards, J. L., Jr., and Kosikowski, F. V. 1969 Electrophoretic proteolytic patterns in Cheddar cheese by rennet and fungal rennets: their significance to international classification of cheese varieties. J. Dairy Sci. 52:1675-1677. Edwards, J. L., Jr. 1969 Bitterness and proteolysis in Cheddar cheese made with animal microbial, or vegetable rennet enzymes. Ph.D. Thesis. Cornell Univ. (Order no. 70-3759) 104 p. Univ. Microfilms. Ann Arbor, Mich. (Diss. Abstr. Int. 30:4194-4195, 1970.) El-Negoumy, A. M. 1968 Starch gel electrophoresis of products of action of crystalline rennin on casein and its com- ponents. J. Dairy Sci. 51:1013-1017. Emmons, D. B., Petrasovits, A., and Gillan, R. H. 1971 Cheddar cheese manufacture with pepsin and rennet. Can. Inst. Food Technol. J. 4:31-37. Emmons 1972 Personal communication. Food Res. Inst., Canada Dept. Agr., Ottawa, Ont., Canada. Ernstrom, C. A. 1961 Milk clotting activity of pepsin and rennin. Milk Prod. J. 52(5):8-11. Fischer, L. 1971 An Introduction to Gel Chromatography. Work, T. S., and Work, E. (edi). lst Ed. Elsevier, New York. 244 pp. Foltman, B. 1970 Prochymosin and chymosin (Prorennin and rennin), pp. 421-436. In: Methods in Enzymology. Vol. 19. Perlmann, G. E., and Lorand, L. (ed.). Academic Press, New York. 1042 pp. plus xvi. 142 Fox, P. F., and Walley, B. F. 1971 Bovine pepsin: Preliminary cheese making experiments. Irish J. Agr. Res. 10:358-360. In: Food Sci. Tech. Abstr. 4(6):l74, 1972. (Original not seen.) Fruton, J. S. 1970 The specificity and mechanism of pepsin action, pp. 401-443. In: Advances Enzymology. Vol. 33. Nord, F. F. (ed.). Interscience Publishers, New York. 595 Funder, S. 1949 Physiological variation in proteolytic properties of molds. XII. Int. Dairy Congr. 2:463-471. 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C. apparatus Corp., Philadelphia, Pa. n.p. 143 Kawai, M., and Mukai, N. 1970 Studies on milk clotting enzymes produced by Basidiomycetes. Part 1. Screening tests of Basidiomycetes for the production of milk clotting enzymes. Agr. Biol. Chem. 34:159-163. Kawai, M. 1970a Studies on milk clotting enzymes produced by Basidiomycetes. Part II. Some properties of Basidiomycete milk clotting enzymes. Agr. Biol. Chem. 34:164-169. Kawai, M. 1971b Studies on milk clotting enzymes produced by Basidomycetes. Part III. Partial purification and some properties of the enzyme produced by Irpex lacteus. Fr. Agr. Biol. Chem. 35:1517- 1525. Kikuchi, T., and Toyoda, S. 1970 Use of microbial rennets in cheese making. XVIII. Int. Dairy Congr. 1E:285. Knight, S. G. 1966 Production of a rennin-like enzyme by molds. Can. J. Microbiol. 12:420-422. Krishnamurti, C. R., and Subrahmanyan, V. 1949 Studies on vegetable rennet. I. The milk coagulating enzyme of Ficus carica. Linn. Preparation and Physico-chemical properties. Indian J. Dairy Sci. 1:27-31. Kyla-Siurola, A-L., and Antila, V. 1970 The proteolysis in cheese caused by microbial rennets. XVIII. Int. Dairy Congr. 1E:283. Labuschagne, J. H., and Jaarsma, J. 1970 Studies on cheese manufactured with microbial rennet. S. Afr. J. Dairy Tech. 2:253-259. Ledford, R. A., Chen, J. H., and Nath, K. R. 1968 Degradation of casein fractions by rennet extract. J. Dairy Sci. 51:792-794. Martley, F. G., Jayashankar, S. R., and Lawrence, R. C. 1970 An improved agar medium for the detection of proteolytic organisms in total bacterial counts. J. Appl. Bact. 33:363-370. 144 Matsubara, H., and Feder, J. 1971 Other bacterial, mold and yeast proteases, pp. 721-63. In: The Enzymes. Vol. 3. Boyer, P. D. (ed.). Academic Press, New York. 886 pp. plus xx. McCullough, J. M., and Whitaker, J. R. 1971 Substrate specificity of the milk-clotting protease from Mucor pusillus determined on the oxidized B-chain of insulin. J. Dairy Sci. 54:1575-1578. McDonald, C. E., and Chen, Lora L. 1965 The Lowry modification of the Folin reagent for determination of proteinase activity. Anal. Chem. 10:175-177. McPhie, P. 1971 Dialysis, pp. 23-32. In: Methods in Enzymology. Vol. 22. Jakoby, W. B. (ed.). Academic Press, New York. 648 pp. plus xv. Means, G. E., and Feeney, R. E. 1971a Modification of groups essential for activity, pp. 24-34. In: Chemical Modification of Proteins. Holden-Day Inc., San Fransico. 254 pp. plus x. Means, G. E., and Feeney, R. E. 1971b Ester- and amide-forming reagents, pp. 139-148. In: Chemical Modification of Proteins. Holden- Day Inc., San Fransico. 254 pp. plus x. Melachouris, N. P., and Tuckey, S. L. 1964 Comparison of the proteolysis produced by rennet extract and the pepsin preparation metroclot during ripening of Cheddar cheese. J. Dairy Sci. 47:1-7. Melachouris, N., and Tuckey, S. L. 1968 Properties of a milk-clotting microbial enzyme. J. Dairy Sci. 51:650-655. Melachouris, N. 1969 Discontinuous gel electrophoresis of whey proteins, casein, and clotting enzymes. J. Dairy Sci. 52:456-459. Meyers, E. and Knight, S. G. 1958 Studies on the nutrition of Penicillium roqueforti. Appl. Microbiol. 6:174-178. 145 Mickelsen, R., and Fish, Nancy L. 1970 Comparing proteolytic action of milk-clotting enzymes on caseins and cheese. J. Dairy Sci. 53:704-710. Morris, T. A., and McKenzie, I. J. 1970 A microbial rennet/calf rennet mixture in Cheddar cheese manufacture. XVIII. Int. Dairy Congr. 1E:293. Motoc, D., and Castin, G. M. . 1970 Study of the action of Penicillium roqueforti. Lucr. Stiint. Inst. Politeh. Galati. 4:231-241. In: Dairy Sci. Abstr. 33:801. (Original not seen.) Nadassky, S. 1972 Sure curd microbial rennet and its suitability for Edam brick cheese manufacture. Prumyl Potravin. 23(2):36-37. In: Food sci. Tech. Abstr. 4(6):l74, 1972. (Original not seen.) Naylor, N. M., Smith, L. W., and Collins, Helen Jo. 1930 The esterase and protease of Penicillium roqueforti. Iowa State College J. Sci. 4: 465-471. Niki, T., Yoshioka, Y., and Ahiko, K. 1966 Proteolytic and lipolytic activities of Penicillium roqueforti isolated from Blue cheese. XVII. Int. Dairy Congr. D:531-537. Nishikawa, I. 1957 Studies on the proteolytic decomposition in cheese. Part II. Characteristics of the proteolytic enzyme of Penicillium roqueforti. Report of Research Laboratory. No. 36. Snow Brand Milk Prod. Co. Ltd., Tokyo, Japan. 9 pp. Organon, N. V. 1970 Purified microbial rennins for use in cheese production. Fr. Pat. 1,592,965. June 26. In: Chem. Abstr. 74:335. (Original not seen.) Oruntaeva, K. B., and Seitov, Z. S. 1971 Isolation, purification, and amino acid compo- sition of rennins A and B from lambs. Biokhimiya. 36:18-21. In: Chem Abstr. 74:36, 1971. (Original not seen.) 146 Osman, H. G., Abdel-Fattah, A. F., Abdel-Samie, M., and 1969a Mabrouk, S. S. Production of a milk-clotting enzyme preparation by Aspergillus niger and the effects of various factors on its activity. J. Gen. Microbiol. 59:125-129. Osman, H. G., Adbel-Fattah, A. F., and Mabrouk, S. S. 1969b Purification and some properties of milk-clotting enzyme from Aspergillus niger. J. Gen. Microbiol. 54:131-135. Proks, J-: Dolezalek, J., and Pech, I. Z. 1956 Biochemical studies about Penicillium roqueforti. XIV. Int. Dairy Congr. 2:401-412. Purkayasta, R., Yaguchi, M., Marier, J. R., and Rose, D. 1967 Distribution of sialic acid among K-casein components. Abstr., J. Dairy Sci. 50:940. Rhodes, C-: Germershausen, J., and Suskind, S. R. 1971 Neurospora crassa: Preparative scale for biochemical studies, pp. 80-86. In: Methods in Enzymology. Vol. 22. Jakoby, W. B. (ed.). Academic Press, New York. 648 pp. plus xv. Richardson, G. H., Nelson, J. H., Lubnow, R. E., and 1967 Schwarberg, R. L. Rennin-like enzyme from Mucor pusillus for cheese manufacture. J. Dairy Sci. 50:1066-1072. Richardson, G. H., and Nelson, J. H. 1968 Rapid evaluation of milk coagulating and flavor producing enzymes for cheese manufacture. J. Dairy Sci. 51:1502-1503. Rickert, W. 1970 The degradation of the B-chain of oxidized insulin by Mucor miehei protease. Compt. Rend. Trav. Lab. Carlsberg. 38(1):1-17. Riehm, J. P., and Scheraga, H. A. 1965 Structural studies of ribonuclease. XVII. A reactive carboxyl group in ribonuclease. Biochem. 4:772-782. Ryle, A. P. 1970 The porcine pepsins and pepsinogens, pp. 316- 336. In: Methods in Enzymolggy. Vol. 19. Perlmann, G. E., and Lorand, L. (ed.). Academic Press, New York. 1042 pp. plus xvi. 147 Sé, F. V. de, and Barbosa, M. 1970a Renneting activity of a vegetable rennet from Cardoon extract (Cynara Cardunculus) compared with animal rennet. XVIII. Int. Dairy Congr. 1E:286. 86, F. V. de, and Barbosa, M. 1970b Comparative rheological behavior of curds from renneting to cutting in cheesemaking using Cardoon (Cynara Cardunculus) and animal rennet. XVIII. Int. Dairy Congr. 1E:287. Sa, F. V. de, and Barbosa, M. 1970c Cheesemaking experiments using a clotting enzyme from Cardoon (Cynara Cardunculus). XVIII. Int. Dairy Congr. 1E:288. Salvadori, P., Bianchi, B., and Cavalli, V. 1962a Differentiation of strains of Pencillium used to manufacture Blue cheese in relation to different proteolytic activity. Latte. 36: 18-22. Salvadori, P., Bianchi, B., and Cavalli, V. 1962b Biochemical studies of 3 strains of Penicillium roqueforti having different proteolytic character- istics and intended for the manufacture of Gorgonzola. XVI. Int. Dairy Congr. B: 455-464. Sannabadthi, S. S., Srinivasan, R. A., and Laxminarayana, H. 1970 Studies on milk clotting enzymes from moulds. XVIII. Int. Dairy. Congr. 1E:278. Sardinas, J. L. 1968 Rennin enzyme of Endothia parasitica. Appl. Microbiol. 16:248-255. Scott, R. 1968 Blue viened cheese. Process Biochem. 3:1-4. Schulz, M. E., and Thomasow, J. 1970 Suitability of milk coagulating enzymes for cheesemaking in relation to cheese variety. XVIII. Int. Dairy Gonr. 1E:321. Shapiro, A. L., Vifiuela, E., and Maizel, J. R., Jr. 1967 Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Comm. 28:815-820. 148 Sherwood, I. R. 1935 The function of pepsin and rennet in the ripening of Cheddar cheese. J. Dairy Sci. 6:407-421. Shovers, J., and Bavisotto, V. S. 1967 Fermentation-derived enzyme substitute for animal rennet. Abstr., J. Dairy Sci. 50:942-943. Srinivasan, M. R., Bhattacharya, D. C., Mathur, O. N., 1970 and Samlik, 0. Studies on the production of soft cheese using different types of rennet. XVIII. Int. Dairy Congr. 1E:322. Stefanowa-Kondratenko, M., Bodurska, I., and Manafowa, N. 1971 The use of an enzyme preparation of Bacillus mesentericus strain 76, in the manufacture of white pickled cheese from sheep and cows' milk. Milchwissenschaft. 26:740-744. Sternberg, M. Z. 1971 Crystalline milk-clotting protease from Mucor miehei and some of its properties. J. Dairy Sc1. 54:159-167. Tam, J. J., and Whitaker, J. R. 1972 Rates and extents of hydrolysis of several caseins by pepsin, rennin, Endothia parasitica protease and Mucor pusillus protease. J. Dairy Sci. 55:1523-1531. Thibodeau, R. and Macy, H. 1942 Growth and enzyme activity of Penicillium roqueforti. Minn. Agr. Expt. Station., Tech. Bull. 152. 56 pp. Thomasow, V. J. 1971a The use of pepsin in cheesemaking. Milchwissen- schaft. 26:276-280. Thomasow, V. J. 1971b Milk clotting enzymes for cheese manufacture. (Transl. from German.) Molkerei-Ztg. Welt d. Milch. 25:470-474. Thompson, V. 1972 Fungal enzyme just like calf rennet. Clots milk for cheese at 45% lower cost. Food processing. 33(10):12. 149 Trop, M., and Pinsky, A. 1971 Mucor pusillus rennin as a synergist to calf rennet. J. Dairy Sci. 54:5-7. Vanderpoorten, R., and Weckx, M. 1972 Breakdown of casein by rennet and microbial milk-clotting enzymes. Neth. Milk Dairy J. 26:47-59. Veringa, H. A. 1961 Rennet substitutes--a review. Dairy Sci. Abstr. 23:197-200. Whitaker, J. R. 1959 Properties of the milk-clotting activity of Ficin. Food Tech. 13:86-92. APPENDIX APPENDIX Type I Medium Martley ep‘gl. (1970) formulated an improved medium for the detection of proteolytic organisms in total bacterial counts. The composition was as follows: 211 Standard Methods Agar (SMA) 23.5 Sodium caseinate 10.0 Trisodium citrate 4.41 Calcium chloride (6H20) 4.38 Type III Medium Czapek-Dox broth was obtained from Difco or formulated as follows: 21./.1 Sucrose 30.0 Sodium nitrite 3.0 Dipotassium phosphate 1.0 Magnesium phosphate 0.5 Potassium chloride 0.5 Ferrous sulfate 0.01 Type IV Medium Meyers and Knight (1958) formulated a medium for the submerged growth of P. roqueforti. The medium con- sisted of: 150 151 U) \ lac Sucrose Ammonium sulfate Dipotassium phosphate Magnesium sulfate (7H20) Potassium chloride Ferrous sulfate (7H20) Sodium acetate Sodium lactate Oleic acid OU'INOOOl—‘KDO I. II I I I NNOOU'IUIODO U'I OD) The oleic acid was eliminated from the above formulation in shake culturing experiments with P. roqpeforti. To one 2 of the above solution, 1.0 m1 of mineral solution was added. On a 100 m1 basis, this solution contained: 23 Cupric chloride 15.5 Zinc sulfate (7H2O) 175.6 Magnesium chloride (4HZO) 36.0 Calcium chloride (ZHZO) 183.4 Ammonium molybdate (4H20) 10.2 Universal Buffer A stock buffer, 0.4 M with respect to phosphate, acetate and borate, was prepared by adding 39.2 g of phosphoric acid, 24.0 g of acetic acid and 24.8 g boric acid to a l 2 volumetric flask (Coch Frugoni, 1957). After solvation of the acidic components, the solution was diluted to volume. A 100 m1 of this stock solution was diluted to 1 Q prior to adjustment of pH (0.2 N sodium hydroxide) and ionic strength (sodium or potassium chloride). The 152 quantity of alkali and salt added to obtained a specific pH and ionic strength given in Table A-1 on following page. Alkaline Copper Sulfate-Sodium Tartrate Solution Protease activity Two m1 of 1.0% copper sulfate (w/v) were mixed with 2 m1 of 2.7% sodium tartrate (w/v). To this, 96 m1 of 0.35 N sodium hydroxide in 2.0% sodium bicarbo- nate (w/v) were added. Protein determination The alkaline copper sulfate-sodium tartrate was prepared as in a except the N of the sodium hydroxide was reduced from 0.35 to 0.20. Ninhydrin Solution Four hundred mg of stannous chloride (ZHZO) were dissolved in 250 m1 of 0.2 M acetate buffer at pH 5.0. This solution was mixed with 250 m1 of methyl cellosolve (Ethylene glycol monomethyl ether) containing 10 g of dissolved ninhydrin (Calibiochem, A grade). The solution was flushed with nitrogen and stored in a brown glass bottle at 4 C. 153 TABLE A-1.-—Protocol for preparation of 0.04 M Universal buffer. . a b g Salt to Attain u-= 0.3C pH DeSlred n 0 NaCl KCl 2 10 0.020 17.35 22.13 3 175 0.036 18.62 23.75 4 235 0.040 18.92 24.13 5 345 0.050 19.71 25.14 5.75 410 0.056 20.03 25.60 6.0 425 0.060 19.92 25.41 7.0 535 0.073 20.23 25.80 8.0 620 0.090 20.00 25.50 9.0 705 0.098 19.83 25.30 10.0 805 0.110 19.76 25.21 11.0 890 0.130 18.23 23.26 12.0 10.75 0.150 17.79 22.70 am1 0.2N NaOH required to bring the pH of l l of 0.04 Universal buffer (pH 1.95) up to the level desired. bThis is the ionic strength of the buffer after "n" m1 of 0.2 N NaOH have been added. cThe g of salt is based on the total volume, i.e., 1000 m1 + n m1 of 0.2 NaOH. 154 Ninhydrin Spray Preparation of this reagent involved solvation of 0.35 g of ninhydrin (Calbiochem, B grade) in 350 ml of absolute ethanol, 135 m1 of glacial acetic acid and 140 m1 of collodin (2, 4, 6 Trimethyl pyridine). Kjeldahl Digestion Mixture Five 9 of copper sulfate (SHZO) and 5.0 g of selenium dioxide were dispersed in 500 m1 of concentrated sulfuric acid. TABLE A-2.--Summary of BP-13 protease properties. Property Value or Comments Optimum pH Stability to pH (maximum) Optimum temperature Stability to temperature Energy of activation for hydrolysis of casein Molecular weight Enzyme type E.C. no. 3.0-BSA; 5.5-casein pH 3.0 to 6.0 45 C (9 min endpoint assay) stable up to 45-46 C for 9 min 8000 cal/mole 45,000 Daltons (Acrylamide gel electrophoresis with SDS) 49,000 Daltons (Gel filtration-G100 Sephadex) Acid protease 3.4.4.99 155 List of MSU photo lab negatives of figures, graphs and photographs in thesis. Figure Negative no. 1 724140-17 2 724140-16 3 724140-19 4 724140-20 5 724140-18 6 724140-3 7 724140—1 8 724140-4 9 724140-6 10 724140-11 11 724140-5 12 724140-12 l3 724140-10 14 724290-3 15 724140-21 16 723625-3 17 723625-6 19 723625-4 20 724290-2 21 724290-4 22 724290-6 23 724140-2 24 724140-9 25 724290-5 26 724140-7 27 724140-14 28 724140-13 29 724140-44 30 724140-15 31 724140-8 32 724290-1 “I111110111111“