3f :\ J3. I .2":- 6.0 conditions metallic ions (Ca2+, Mg" ) react with protein polyacrylate and form calcium or magnesium polyacrylate salts. In the meantime the enzyme is solubilized and released from precipitate to solution. Generally, the recovery of enzyme is more than 80%. 3. Recovery of polyacrylic acid from calcium or magnesium polyacrylate can be achieved by mixing with 2N H2804. H2804 will regenerate water soluble polyacrylic acid. In some cases the regeneration of polyacrylic acid is up to 90%. The purification is based on the selectivity of the reaction of proteins with polyacrylic acid. Formation of an insoluble protein-polyacrylate complex is dependent on the affinity of particular proteins for polyacrylic acid, the molecular weight of polyacrylic acid, and the ionic strength and pH of the solution. Another advantage with polyacrylic acid at pH <6.0 is that mono- and polysaccharides, amino acids, oligopeptides, lipids, nucleotides, nucleic acids and inorganic salts do not form precipitates with polyacrylic acid (Stemberg, 1976). 22 2.2.1.7 NON-IONIC POLYMER PRECIPITATION AND TWO-PHASE PARTITIONING Relatively few reports have appeared in the literature on the precipitation of proteins by nonionic high molecular weight polymers (Janssen and Ruelius (1968), Fried and Chun (1971), Foster gt 11; (1973), and Albertsson (1986)). The mechanism of precipitation is not fully understood, but the phenomenon is closely related to the formation of liquid-liquid two phase systems from mixtures of aqueous polymers. The most commonly used two-phase systems are higher molecular weight polymers dextran and polyethylene glycol (PEG) (Kula (1979), Hustedt gt a1. (1978) and Kroner (1982)). However, other polymers including methylcellulose, polyvinyl alcohol, and ficol (Albertson, 1986) have been used to form aqueous two-phase systems. Each of the above applications is based on the powerful influence of the polymer on the interaction of the protein with its aqueous environment. The partition is based on the principle that aqueous solutions of a mixture of two neutral polymers will form two phases, and each protein in this solution will have a unique distribution coefficient. The partition of a compound in aqueous two-phase systems is mainly influenced by molecular weight, temperature, pH, ionic strength and the types of ions included in the system but it does not depend on the concentration of the desired product (Kroner _e_t_ _al. (1982) and, Mattiasson and Kaul, 1986). Unfortunately, the conditions for favorable partition have to be found empirically for the enzyme of interest. The main advantage of using the two-phase dextran-PEG system in protein purifications is its non-toxicity and resulting suitability for use in pharmaceuticals and safe consumption; but the high cost limits its application. 23 2.2.2 CHROMATOGRAPHIC METHODS Chromatographic enzyme purification methods are the most effective of all the separation methods. The mechanism of the separation will depend in different cases on adsorption, ion exchange, specific affinity to immobilized ligands, and molecular sieving effects. The basic principles of chromatographic methods and the use of available ion exchange, gel chromatography and affinity chromatography media have been described by Hirnmelhock (1971), Cuatrecases and Anfiusen (1971), Cooper (1977), Kremmer and Boross (1979), and from the manufactures of the media (Pharmacia Fine Chemicals, Bio-Rad Laboratories, LKB Produckter Ltd., Whatrnan Biochemicals Ltd., Arrricon Corporation and others). 2.2.2.1 NON-SPECIFIC ADSORPTION CHROMATOGRAPHY Purification is performed by using substances that adsorb the proteins by Van der Waals forces and stearic interactions. These forces are most important for substances with few polar residues. Only a few non-specific absorbents are important in protein isolation. Calcium phosphate gel, particularly in its crystalline form hydroxyapatite (HA), has been widely used on a laboratory scale. Although the mechanism by which HA separates proteins is not fully understood, it has been suggested that calcium and phosphate ions on the surface of HA crystals participate in the interactions with charged groups of protein amino acids under defined conditions of pH, temperature, type of ions, and ionic strength. Acidic and neutral proteins bind to calcium sites on HA, and the elution of these proteins is 24 achieved with low concentration of phosphate buffers at about pH 6.8. Basic proteins, however bind to phosphate groups on HA crystals with adsorption and elution being strongly affected by the presence of NaCl, KCl and CaClz. In enzyme purification procedures, HA is generally used as a final clean-up methods following other purification steps. Other non-specific absorbents that have been used are gamma-alumina gel and diatomaceous earth. As examples, alkaline phosphatase from bacteria was purified by using HA (Y eh and Trela, 1976) and diatomaceous earth (celite) is used in the purification of two beta-lactamases from B. census (Wang e_t aL 1979). 2.2.2.2 ION EXCHANGE CHROMATOGRAPHY Ion-exchange chromatography is the most preferable and most widely used methods for industrial-scale protein purification because it can be easily scaled up by converting from a column technique to a batch process. It can be defined as the separation of one species from another, applied to the mobile phase, by the differential binding and release of these solutes to the fixed charges of the ion exchanger. Ion-exchange chromatography of enzymes usually employs derivatives of cellulose, agarose, dextrans or resins. The cationic exchangers used for enzyme purification bear negatively charged groups attached to the matrix material, the pK values of these groups are in the range of 2-7. The anion exchangers most commonly used are diethylamino-ethyl (DEAE) cellulose, triethylamino-ethyl cellulose, and ECTEOLA cellulose; the basic substituents of the celluloses have pK values in the range of 7-10. 25 The separation of proteins by ion exchange chromatography is obtained by reversible adsorption. Most of the experiments are performed in two stages. The first stage is sample application and adsorption. Unbound substances can be washed from the exchanger. In the second stage, enzymes of interest are eluted from the column and collected in separate fractions. The fractionation is achieved since various proteins have different affinities for the ion-exchanger due to the differences in their charge. However, the net charge, charge density and molecular size of the protein as well as pH and ionic strength of the solution are all parameters which affect the separation of enzymes by ion exchange chromatography. Proteins are not bound to any type of ion-exchanger at its isoelectric point (IEP). Protein will bind to the exchangers when electrostatic interactions between protein and exchangers are strong. At this point, the charge on proteins is high and opposite to that of the exchanger. Normally, a cation exchanger should be used if the protein of interest is most stable below its IEP and an anion exchanger should be used if the protein is most stable above its IEP. The pH for binding should be at least one pH unity above or below the IEP’s of proteins in order to give a high ionic charge on both the exchanger and the proteins. In ion exchange chromatography one can choose whether to bind the enzymes of interest or to adsorb impurities and allow the substance of interest to pass through the column. Generally, it is more useful to adsorb the substance of interest, since this allows a greater degree of purification. A general reference for experimental details of ion exchange chromatography is the paper by Himmelhoch (1971). 26 2.2.2.3 GEL FILTRATION This separation technique partitions two aqueous phases of identical composition on the basis of molecular size in a column or slab. One phase is freely flowing, the other extrapped by a gel maze in a beaded form. The sample is applied on the surface of the column of appropriate porous beads of the hydrated gel and solvent is percolated through the column. Large molecules which cannot diffuse into the porous structure of the beads, are excluded and pass through in the void volume of the column. Small molecules elute only when a volume equal to the total water content has passed through the column. Intermediate size molecules elute somewhere between the larger and smaller molecules according to their K“ value. These intermediate size molecules do not become permanently trapped in the column if no other factors intervene. Separation occurs according to size, and the molecular weight can be estimated if calibrating proteins are run as references. Generally K,r (defined as VMOJVW“) is plotted against log M, (molecular weight) giving a selectivity curve for the particular gel and column. A wide variety of beaded gels is available: cross-linked dextrin (sephadex), polyacrylamide gel (sephacryl, Bio-Gel P), agarose (sepharose, Bio-Gel A) and cross-linked a gamse (sepharose CL). Together, they cover intermediate-size molecules (M,) ranging from 500 to 10°. In all of these gels the void volume is approximately 1/3 of the column volume and is determined by measuring the elution volume of an excluded substance like Blue Dextrin 200. Much effort has been exerted to determine the molecular weight of globular proteins and their behavior in Sephadex or Agar-gel columns (Andrews, 1964). Agar 27 gels can be used to separate proteins over very wide molecular weight ranges. Over small ranges, much better separation can be obtained with gel filtration media composed of cross-linked dextrins or polyacrylamide. Therefore, a more critical appraisal of the relationship between the molecular weight of proteins and their gel-filtration behavior is possible with these media than with agar gels. The gel filtration chromatography technique is presented by Kremmer and Boross (1979), Delaney (1980) has reviewed industrial GFC of proteins. With respect to large scale use, GFC has been applied to the removal of lipase from microbial rennet (Somkuti, 1974). 2.2.2.4 AFFINITY CHROMATOGRAPHY Affinity chromatography occupies a unique place in separation technology which relies not on the general physicochemical pr0perties of a molecule, but rather on the presence of very specific biological and/or chemical functions in the molecule to be separated. In practice, this separation involves the following steps: 1) a solution with desired compound is passed through a column containing a highly specific ligand immobilized on a solid support; 2) as the fluid passes through the column, the desired component binds selectively, and reversibly to the ligand with most impurities passing unhindered; 3) any residual impurities are removed by flushing the column with an appropriate buffer solution; and 4) the compound now purified but still bound, is then recovered by passing through the column a solution that disrupts the ligand binding interaction by changing ionic strength or pH, for instance. 28 The solid support on which the ligand is immobilized should posses the following properties: 1. The matrix must form a loose porous network which permits unimpaired movement of large macromolecules. The gel particles should be uniform, spherical, and rigid with good flow properties. The matrix should not interact with proteins in general in order to avoid non-specific adsorption. The inner support must have abundant supply of chemical groups which can be activated or functionalized to allow the covalent attachment of a variety of ligands. The gel must be mechanically and chemically stable to the conditions of coupling, adsorption, and elution. The most widely used solid supports are hydrophilic cellulose derivatives, polystyrene gels, cross-linked dextrans, beaded agarose, glass beads and polyacrylamide gels and ligands are antibodies, antigens, enzyme inhibitors, isolated receptors, and more recently, closed receptors (Bailow e_t al, 1987). The preparation of a number of chemical derivatives of agarose and polyacrylamide gel bead have been described by Cautrecases and Anfiusen (1971). Dye-ligand chromatography, hydrophobic interaction chromatography, and irnmunoaffinity chromatography are similar to affinity chromatography. All these techniques have been used widely for fractionation and purification of proteins and are discussed in turn. 29 a. Dye-ligand chromatography; Textile dyes are used as general ligands and these dye-ligand media contain a mono- or dichloro substituted triazinyl group, and are sold as Cibacron dyes or Procion dyes. These dyes can be readily immobilized on agarose or other gels to form general purpose affinity matrices. Furthermore, these dyes are cheap, readily soluble, stable, and readily available. Since these are readily available and cheap, this method has been widely used in the industrial and large scale protein purification. b. Hvd_ronhobic interaction chromatography, A type of affinity chromatography in which the affinity arises through the hydrophobic interactions of the apolar ligands attached to the chromatographic carrier (usually agarose) and the hydrophobic regions of the proteins to be absorbed. The extent of these hydrophobic interactions depends on factors such as the length of the immobilized hydrophobic chains, ionic strength of the buffer, nature of the salts in the buffer, pH and temperature of the buffer, presence of polarity—reducing agents, and presence of a detergent (Rosengren e_t a_l. 1975 and Jennissen and Heilmeyer, 1975). For further references, reviews by Miles laboratories (1979) and by Pharmica Fine Chemicals (1982) are available. This technique has also been used for the large scale purification of glucocerebroside B—glucosidase from human placenta (Pharmica, 1982). c. Immuno affinity chromatogr_aphy; This chromatography exploits the unique specificity, high affinity, and reversibility of the antibody-antigen interactions. In this technique, the desired antigen is adsorbed to the antibody which has already been immobilized by covalent linkage to an insoluble matrix, 30 and then recovered by washing with an agent that disrupts the immune complex. Conversely, the antigen may be immobilized and used to purify its respective antibody. A great many applications of the technique have been described along with a number of review articles (Robbins and Schneerson, 1974, Eveleigh and Levy 1977, Fuchs and Sela, 1978, Krisfiansen 1978, Hudson and Hay 1980, and Eveleigh 1982). In early work only polyclonal antibodies were used to purify antigens, but with the advent of the hybridoma technique, monoclonal antibodies have become available. Monoclonal antibodies have three main advantages over polyclonal antibodies. 1. Highly purified antigen is not required for immunization. 2. Monoclonal antibodies are directed against a single epitope. 3. Monoclonal antibodies can be produced in unlimited quantities. Since monoclonal antibodies can be produced in large quantities this technique is also attractive for the purification of antigens on an industrial scale. d. Affinity precipitation; Affinity precipitation uses the solubility of a ligand protein complex for separation. Two options are available, one in which the complex is insoluble upon formation; and the other in which the solution buffer must be changed to cause it to precipitate (Flygare e_t a1” 1983). 2.3 ULTRAFILTRATION Ultrafiltration (UF) is a pressure driven membrane separation process for dissolved and suspended materials according to the molecular weight and size. Substances 31 smaller than the pore size of the membrane will pass through with the solvent while larger particles, are retained. In general, UF membranes can separate species ranging from molecular weight 550 to 1,000,000 daltons and the pore size of the membrane ranges from 10 to 100 angstroms. UP is a very simple procedure and requires no phase change, no chemical addition, and less energy, time and cost. Recent advances in membrane technology and new developments in biotechnology have made UP the most useful method in bridging the gap between laboratory scale and industrial scale set ups (Underkofler (1976) and Blatt (1971)). In addition, this method has been used to replace and/or supplement many conventional separation techniques, such as centrifugation. dialysis, distillation, salt precipitation, lyophilization, evaporation, and chromatography Presently, the need for the extensive isolation and concentration of recombinant DNA products and monoclonal antibodies show that the UP concentration process is an essential separation process for the pharmaceutical, chemical and food processing industries. There are three basic configurations of UP membranes available. They are hollow-fiber, plate and frame and spiral cartridges. Hollow fiber membranes, which are supplied in self-contained cartridge housings, are easy to clean and allow good product recovery, but they are somewhat limited in pressure capability. On the other hand, plate and frame membranes are capable for high pressure operation, but can have cleaning and product recovery problems. Traditionally cellulose membranes are the most common for membrane separation (Reid and Breton, 1959). These membranes exhibit remarkable selectivity between salts and water, but suffer chemical and biological attack and have low resistance to 32 temperature. Invention of synthetic polymer membranes which are physically and chemically more rugged and durable, replaced these cellulose membranes. Most commonly used synthetic membranes are aromatic amides, polycarbonates, polyacrylonitrile-polyvinyl chloride copolymer and polysulfone. Most recently, composite membranes also been used in membrane separation. The basic problemiin membrane separation is the interaction between the solutes and the membrane itself. This can decrease the flux significantly. It can be overcome either by back flushing with cleaning solution or using a membrane that has no affinity for the solutes or the membrane surfaces (Le and Howell, 1983). The application of UP can be categorized into three groups: concentration, diafiltration, or purification. In industry, UF also been used in the following ways: 1. To concentrate and diafiltrate of final product generally after gel filtration, thus substantially reducing volume prior to lyophilization. 2. To concentrate the product prior to salt precipitation, thereby reducing the amount of salt required. The smaller volumes are easier to separate after precipitation and salt disposal expenses are reduced. 3. To remove such low molecular weight contaminants as peptides, amino acids, sugars, and salts by initial diafiltration. 4. To remove alcohol by following solvent precipitation and redilution of the precipitate. In this project UP is used to remove the cells, concentrate fermentation broth, and concentrate the product prior to another purification step. 33 2.4 GEL ELECTROPHORESIS Electrophoresis, an enzyme purification technique based on the size and charge of the molecule, provides for high resolution of purification and is capable of purifying very minute quantities. This method enjoyed some p0pularity for enzyme separation for quite a few years but it is now largely used as a tool for separation of isozymes and other enzymes related diagnostic purposes like molecular weight determination. Polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing are the techniques frequently used for laboratory scale enzyme purification and characterization. Polyacrylamide is the most common support matrix for the electrophoretic separation of proteins subunits (Southern, 1979 and Saeed and Boyde, 1980). However, other support media, such as Bio-Gel P-300 (Saeed and Boyde, 1980), can also be used. Only 10-200 micrograms are typically separated by analytical PAGE. The fractionation of larger quantities (up to 250 mg) by preoperative PAGE requires special equipment (Southern, 1979). Recently, an industrial scale continuous electrophoretic separator has been developed by the biochemistry group at Harwell Division of the UK. Atomic Energy Authority (Didcot, England). This unit can draw off as many as 29 separate fractions in potential applications as isolation and purification of high value pharmaceutical enzymes and other products. PAGE has become a common and important tool for the molecular biologist to determine the size of macromolecules, DNA, RNA, and protein. The procedure and the analysis are simple. Several methods have been developed for the estimation of the proteins using ionic detergents and solvents. Lambin pt g. (1976) demonstrated 34 that electrophoresis in linear polyacrylamide gradient gels, combined with sodium dodecyl sulfate (SDS) treatment, can be successfully used to estimate the molecular weight (M,) of proteins. Using only one electrophoretic step, this technique provides accurate measurement for proteins or glycoproteins with M,’s between 10‘ and 10‘. In the case of glycoproteins, this estimate is not modified by the carbohydrate content (Lambin and Fine, 1978). In this project the molecular weight of the B—amylase is determined by PAGE-SDS method and compared. For further details many review articles are available by Shaw (1969), Omstein (1964), Davis (1964), Shuster (1971) and Smith (1967). 35 LITERATURE CITED Albertsson, P. A, Partition of Cell Pa_rticles and Macromolecules, 3rd (ed.,) Wiley-Interscience, New York, 1986. Andrews, P., "The gel-filtration behaviour of proteins related to their molecular weights over a wide range", Biochem. J., 1965, 96:595-606. 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Anfiusen, "Affinity Chromatography", Methods Enzymol., 1971, 22:345. Davis, B. "Disc Electrophoresis-II: Methods and application of human serum proteins", J. Annals, N. Y. Acad. Sci, 1964, 121:404-444. Delaney, R. A. M., In: Applied Protein Chem. R. A. Grant, (ed.), Applied Science Publishers, U. K., 1980, 233-80. Eveleigh, J. W. and D. E. Levy, "Immunochemical characteristics and preparative application of agarose-based irnmunosorbents", J. Solid Phase Biochem., 1977, 2, 1:45- 78. Eveleigh, J. W., In: Affinity Chromatoggaphy and Related technigues (T. C. J. Bribnan, J. Visser and R. J. F. Nivard, ed.), Elsevier Scientific Publishing Company, Amsterdam, 1982, 293. Flygare, S., T. Griffin, P. Lawson, and K. Mosback, "Affinity precipitation of dehydrogenases", Anal. Biochem., 1983, 133, 2:409-16. Fogarty, W. M., and C. T. Kelly, "Amylases, amyloglussidases and related glucanases", In: A. H. Rose (ed.), Microbial enzymes mid bioconversions, Academic Press, Inc., 1980, 115- 169. 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Phillips, In: Handbook of Enzyme Biotechnology, A. Wiseman, (ed.), Ellis Horwood, Chichester, U. K., 1975, 58-88 and 181-202. Meyer, K. H., P. F. Spahr and E. H. Fisher, "Purification cristallisation et proprietes de la B-amylase de ble", Helvvetica Chimca Acta, 1953, 36:1924-36. Momsen, W. E. and H. L. Brockman, "Purification and characterization of cholesterol esterase from porcine pancreas", Biochem, BiOphys. Acta 1977, 486:103-13. Morris, C. I. O. R. and P. Morris, Separation Methods in Biochemisg, Pitman, Publ. Ltd., London, Engl., 1976, 834. 39 Ng, T. K., and J. G. Zeikus, "Comparison of extracellular cellulase activities of clostridium therrnolceeum LQRI and Trichoderrna viride QM9414.", Appl. Environ. Microbiol., 1981, 42:231-40. Nummi, M., R. Vilhunen and T. M. Enari, "Exclusion chromatography of barley B- amylase on sephadex G-75", Acta Chem. Scand., 1965, 19:1793-5. Okon, E. U. and A. O. Uwaifo, "Particl Purification and Properties of b-amylase Isolated from Sorglium Bicolor (L.) Moench", J. Agric. Food. Chem., 1984, 32:11-14. Omstein, L., "Disc electrophoresis, background and theory ", Annals, NY. Acad. Sci., 1964, 121:321-49. Reid, C. E. and E. J. Bretow, "Water and ion flow across cellulosic membranes", J. Appl. Polym. Sci., 1959, 1:133-43. Robbins, J. B. and R. Schneerson, "Immunoadsorbents", Methods in Enzymology (W. B. Jakoby and M. Wilchek, eds.), Academic Press, 1974, 36:703. Rbbyt, J. F., and W. J. Whelan, "The B—amylase", In: J. A. Radley (ed.), Starch and its derivatives, Chapman and Hall Ltd., 1968, p. 477-497. Rosengren, J., S. Pahlrnan, M. Glad and S. Hjerten, "Hydrophobic interaction chromatography on non charged sepharose derivatives", Biochim. Biophys. Acta, 1975, 412:51-61. Saeed, S. A. and T. R. C. Boyde, "Preparative gel electrophoresis at high sample load. The effect of some experimental variables on separation performance", Prep. Biochem., 1980, 10, 4:445-62. Schink, B., and J. G. Zeikus, "Clostridium thermosulfurogenes sp. nov., a new therrnophile that produces elemental sulphur from thiosulphate", J. Gen. Microbiol., 1983, 129:1149-58. Shaw, D. J., Electrophoresis, Academic Press, London, N.Y., 1969. Shuster, L., "Preparative Acrylamide Gel Electrophoresis: Continuous and Disc Techniques", Methods in Enzymol, 1971, 22:412. Smith, I., "Ehromatographic and electrophoretic techniques", Vol. I, Electrophoresis is Stabilizing Media, Academic Press, New York, 1967. Somkuti, G. A., "Control of lipase content in mucor rennet", J. Dairy Sci., 1974, 57(8):898-9. 40 Southern, E. M., "Measurement of of DNA length by electrophoresis", Anal. Biochem., 1979, 100:304-18. Stemberg, M. and D. Hershberger, "Separation of proteins with polyacrylic acids", Biochem. Biophys. Acta, 1974, 342:195-206. Stemberg, M., J. P. Chiang and N. J. Eberta, "Cheese whey protein isolated with Polyacrylic acid", J. Dairy Sci., 1976, 59:1042-50. Swimmer, S., In: Source Book for Food Enzymology, The AVI Publ. Co., Inc., Westport. Conn., 1981, pp. 105-122. Takasaki, Y., "Productions and utilization of B—amylase and pullulaurase from Bacillus cereus var. mycoides.", Agri. Biol. Chem, 1976a, 40:1515-22. Underkofler, L. A., "Microbiol enzymes", Ind. Microbiol, 1976, 128-64. Wang, D. I. C., C. L. Cooney, A. L. Demain, P. Dunnill, A. F. Humphrey, and M. D. Lilly, in Fermentation afl Enzyme Technology, Wiley (Interscience), New York, 1979, 238-310. Wheelwright, M., "Designing downstream processes for large-scale protein purification", Bio/Technology, 1987, 52789-93. Wolnak, B., and associates, "Present and future technological and commercial status", report prepared for the National Science Foundation, U. S. Dept. of Commerce Natl. Tech. Inf. Serv. Doc. PB-219 636, December, 1972. Yeh, M. and J. M. Trela, "Purification and characterization of a repressible alkaline phosphatase from Thennus aguaticus", J. Biol. Chem, 1976 251, 10:3134-39. Zeikus, J. G, "Thermophilic bacteria: ecology, physiology and technology", Enzyme Microb. Technol., 1979, l .-243 52. CHAPTER 3 FERMENTATION AND CONCENTRATION OF A NOVEL THERMOSTABLE EXTRACELLULAR B—AMYLASE FROM CLOSTRIDIUM THERMOSULFUROGENES 3.1 ABSTRACT An thermostable extracellular enzyme, B-amylase was produced from Clostridium thermosulfurogenes with different carbon sources. Different sizes of fermenters were used and the fermentation time was optimized. Neither the size of the ferrnenter nor the fermentation carbon sources influenced the final total B—amylase activity or the fermentation time for the production of B—amylase from C.thermosulfurogenes. Concentration by ultrafiltration showed higher B-amylase activity recovery and purification due to the removal of peptides and small proteins with permeate. B-amylase produced by fermenting maltrin yields higher [Iamylase activity recovery with 100,000 mwco UF membrane. On the other hand, B-amylase produced by fermenting maltose yields very poor recovery with the same membrane. It concluded that the residual maltrin forms a larger molecular weight complex with the B-amylase resulting in an increase in apparent molecular weight. 3.2 INTRODUCIION Amylolytic enzymes are essential for a number of industrial processes, mainly in saccharification of starch and malting in beer production. The main arnylolytic 41 42 enzymes used in the industry are a-amylase, B-amylase, glucoamylase, and pullulanase. A premium is placed on thennostability and thermoactivity of the above enzymes used in bioprocessing of starch. However, most of the microbial amylases used in industry are not thermostable and not active at higher temperature. It has been suggested that thermoanaerobes have the potential for the production of active and thennostable enzymes (Zeikus, 1979). The enzyme produced and partially purified in this project is an extracellular thennostable B-amylase (MBI B—amylase) developed from the thermoanaerobe C_.tfhennosglfurogenes at the Michigan Biotechnology Institute (MBI). 3.2.1 B—AMYLASE B—amylase (EC 3.2.1.2, a-l,4-D-Glucan maltohydrolase) is an exo-acting carbohydrase which cleaves a-l,4 glucosidic linkages from the non-reducing end of a starch molecule and generates maltose as a major final product. Since B—amylase cannot react or bypass the branching points of the carbohydrate chain, the hydrolysis stops near the branching points, thus yielding maltose and B-amylase limit dextrin (IS-limit dextrin). B—amylases are well studied and characterized in higher plants and have also been reported in micro-organisms (Robyt and Whelan, 1968, Higashihara _e_t g, 1974, Shinke _e_t a1, 1975, Takasaki, 1976, and Thomas pt a_l., 1980 ). However, most of the B-amylases are not thennostable and not active at higher temperatures. The MBI B-amylase of the current study, is active at or above 75°C (Hyun and Zeikus, 1985). This enzyme has a wide pH stability between 3.5 to 7.0 with an optimum pH at 5.0. The general mechanism for regulation of B—amylase synthesis, 43 and its ability to bind starch and to produce maltose syrups have been studied and reported by Hyun and Zeikus (1985) and Saha _e_t, pl, (1987). In a previous study, B-amylase produced from fermentation of maltose was purified and characterized at MBI (Shen gt 5;, 1988). The purpose of the present work was to study the effect of different carbon sources on the B-amylase fmal activity during fermentation and initial concentration by ultrafiltration (UF) membranes. Iii-amylase was produced in different sizes of batch fermenters using different substances (maltose, maltrin, and glucose) to analyze a simple scale-up procedure. 3.2.2 ULTRAFILTRATION Ultrafiltration is a pressure driven membrane separation technique based on molecular weight and size. Almost every bioseparation process uses UF to remove the cells, to clarify the whole broth, and to concentrate the bio-products from invariably dilute and complex aqueous mixtures. This is a low cost, short processing time unit operation which results in high product recovery. UF was used to remove the cells and to concentrate the fermentation product in the present study. Spiral and hollow fiber membranes with different pore sizes were used to concentrate B—amylase produced by fermentation of different carbon sources (substrates). 3.3 MATERIAL AND METHODS 3.3.1 ORGANISM AND FERMENTATION The catabolic repression resistant mutant H-12-1 (Hyun and Zeikus, 1985b) 44 derived from C.thermosulfurogenes wild strain 48 (ATCC 33743) was used. The culture was grown at 60°C in 26 ml anaerobic pressure tubes (Bello Orlans, Inc. Vineland, NJ.) that contained 10 ml of TYE (trypticase and yeast-extract) medium supplemented with 0.5% soluble starch and Nz-CO2 (95:5) gas head space. B-amylase was produced in TYE or corn steep liquor medium with different carbon sources. The components of the lab-medium are listed in Table 3.1. For simple scale-up, two different Bruan fermenters (14 and 120 liters) and a 300 liter Lab-Line fennenter were used to produce B-amylase. All the components were proportionally increased according to the fermentation volume. Initially, the seed culture was expanded to 500 ml in a round bottom flask and then was transferred anaerobically to the large fermenters. Ferrnenters were stirred at a constant rate (400 rpm.) and Nz-CO2 (95:5) gas was purged for a few hours to remove the dissolved oxygen. B-amylase was produced anaerobically at 60°C with a slightly higher pressure than the atmosphere. An anti-foarning agent was added to avoid excessive foaming caused by high production of gases during fermentation. The turbidity of the culture was measured . hourly and the fermentation was stopped after the exponential growth period of the enzyme. The culture was allowed to cool to ~ 15°C before concentration. 3.3.2 ENZYME ACTIVITY AND PROTEIN ASSAYS Enzyme activity was assayed by incubation of the reaction mixture (1 ml) which consisted of 2% soluble starch, acetate buffer (50 mM, pH 6.0 with 5mM CaClz), and proportionally diluted enzymes at 60°C, for 30 minutes. The reducing sugars released by B-amylase action on starch were estimated by the DNS (dinitrosalicylic acid) 45 TABLE 3.1 COMPOSITION OF TRYPTICASE AND YEAST-EXTRACT MEDIA USED FOR THE PRODUCTION OF B-AMYLASE Components Per liter basis Yeast 3 g Trypticase 10 g Salts 3.6 g Carbon source 10 g Vitamins 10 g Trace elements 10 ml 46 method (Miller, 1969). One unit of enzyme activity is defined as a micromole of maltose released per unit time under the conditions of the assay. Total protein concentration was estimated using the Lowry e_t _a_l., (1957) assay method with bovine serum albumin as a standard. 3.3.3 ULTRAFILTRATION Concentration studies were conducted by UF before and after removal of cells by centrifugation (12,000 rpm, 30 min. at 4°C). These experiments showed that there is not a significant difference in recovery if centrifugation precedes UF. Therefore B-amylase was concentrated and cells removed by UP and centrifugation, respectivelly. B—amylase produced with different carbon sources was initially concentrated by UF with 30,000 and 100,000 MW CO diaflo hollow fiber membranes (Amieon) and a 30,000 MWCO spiral membrane (Amicon). Recirculation rate was varied for different size membranes but was constant for each configuration. Membranes were back flushed at a different time intervals to avoid fouling. [i-amylase activity and total protein concentration were assayed before and after each filtration study. 3.4 RESULTS AND DISCUSSION 3.4.1 FERMENTATION B-amylase was produced more than twenty times in all three fermenters. 47 Depending on the activity of the inoculated culture, the fermentation time varied from 8 to 12 hours. In a few instances, when B-amylase activity or the turbidity of the media did not increase after inoculation, media were reinoculated with a fresh seed culture and successfully harvested. A typical plot for the change in turbidity with time for B-amylase produced with maltose and maltrin is shown in Figure 3.1. Final B-amylase activity was assayed in each fermentation, and it was not influenced by the size of the fermenter. B-amylase activities with different carbon sources are shown below. Carbon Source A660 B-amylase Aetivity (activity unit) 1% Dextrins 2.6 30 to 40 1% Maltose 2.3 30 to 44 1% Glucose 0.8 20 to 35 These results also showed that there were no mass transfer problems during scale-up. The maximum activity obtained was 45 U/ml in a 120 liter fermenter with 1% maltose. Fermentation results show that a growing seed culture is essential for the production of B—amylase. This would exclude the possibility of B—amylase produced from its substrate (Ii-amylase could be produced from plant tissues also) and confirmed that the B-amylase is produced only from C. thermosulfurogenes. The fermentation time neither depended on the size of the fermenter nor the carbon source 48 40. 3- Tl‘l‘l‘T‘l‘l‘U‘I'lTITTPI'1‘7‘ 2.— (l) - O C 3‘ O . ._ .0 ._>_ L. +1 O . O (I) <1 .0 < u 1... 0—0 glucose H maltose _ o—o maltrin O r TUTTITTTTI‘I‘T‘I‘FFI‘I‘ - O. 1. 2. 3. 4. 5. 6. 7. 8. 9.10.11.12.13.14.15. Time (Hrs) Figure 3.1. B-amylase activity and turbidity (absorbance) history during prodoction of B-amylase with three different carbon sources glucose, maltose and maltrin. ------ absorbance, ...... B-amylase activity 49 (substrate) of the media, but it was only influenced by the activeness of the seed culture. Seeding a fermenter with culture at its exponential growth would reduce the fermentation time a great deal. Although the final turbidity was low in glucose fermented [S-amylase, the activity was not influenced by the size of the fermenter or the fermentation carbon source. The turbidity increase was due to production of more cells without increasing the B-amylase activity. 3.4.2 ULTRAFILTRATION Since there was no influence in B—amylase recovery for removal of cells before concentration, B-amylase produced by fermenting different carbon sources was concentrated with cells by UF. Two series of experiments were then conducted: 1. B-amylase produced by fermenting four different carbon sources was concentrated with a 30,000 MWCO spiral membrane and an average permeation rate of 500 ml/min through the membrane was maintained in each run. In all runs more than 85% of B-amylase activity recovery with ten fold purification was achieved (Table 3.2). There were no significant fouling problems or drop in permeation rate due to different carbon sources. Significant increases in specific activity were due to the removal of small peptides and proteins with the permeate. Fermentation carbon sources showed no influence in the recovery or in the purification fold of B-amylase with 30,000 MWCO membrane. 2. B-amylase produced with dextrins and maltose was concentrated with 30,000 and 100,000 MWCO hollow fiber membranes. Results are shown in Table 3.3. The highest purification and recovery for B-amylase with dextrins was 50 TABLE 3.2 INITIAL CONCENTRATION OF B—AMYLASE FERMENTATION BROTH WITH CELLS (WITH FOUR DIFFERENT CARBON SOURCES) BY A 30.000 MWCO SPIRAL MEMBRANE. B—amylase Carbon Volume Specific Recovery source (ml) activity (%) (U/mg) original maltose 94,000 10.0 100 concentrated 6,000 141.1 88 original soluble 1 1,000 6.0 100 concentrated starch 1,200 62.8 89 original maltrin-40 10,000 8.5 100 concentrated 840 88.3 88 original maltrin-100 8.500 6.6 100 concentrated 740 75.5 85 51 TABLE 3.3 CONCENTRATION OF B-AMYL_ASE BY 30,000 AND 100.000 MWCO DIAFLO HOLLOW FIBER MEMBRANES AS A FUNCTION OF CARBON we B—amylase Carbon Membrane Specific ’ Recovery source cut off activity (%) (MW CO) (U/mg) original maltrin 100,000 56.0' 100 concentrated 248.2 93 original maltrin 30,000 57.3' 100 concentrated 149.5 85 original maltose 100,000 10.5 100 concentrated 50.0 51 original maltose 30,000 10.0 100 concentrated 141.0 88 ’ higher specific activity fl-amylase was obtained from recycle cells experiments by Dr. A. Nipkow at MBI. 52 obtained by the 100,000 MWCO UF membrane but the same membrane resulted in very poor recovery and purification for B-amylase produced by fermenting maltose. This suggests that particles formed in dextrins fermented B-amylase are larger or form a more stable aggregate than in those formed in maltose. The starch dextrins may form a biospecific complex with B-amylase or help to form stable B—amylase aggregates. Using a larger MWCO UF membrane, reduces the purification cost and time for dextrin fermented B-amylase. The molecular weights of the B-amylases produced with maltose and dextrin were estimated by gel filtration chromatography (results are shown chapter 4). B—amylase produced from dextrins showed a portion of B-amylase activity at higher molecular weight, and the remainder is at a lower molecular weight similar to the maltose derived case. The effective higher molecular weight may be due to the aggregation of B-amylase subunits or binding of B-amylase to dextrin thereby forming a complex. 3.5 CONCLUSIONS 1. Production time of B-amylase in a batch fermenter requires eight to twelve hours, and could be reduced by inoculating with a seed culture at its exponential growth stage. 2. Final B-amylase activity during fermentation does not depend on the size of the fermenter or the fermentation carbon source (substrate) for the range of fermenter sizes studied. 3. Concentration of B-amylase with 30,000 MWCO UF membrane gives 85% 53 activity recovery with ten fold purification of B-amylase from all four carbon sources considered. . A 100,000 MWCO UF membrane recovers approximately 90% activity of B-amylase produced by maltrin fermentation and only 50% activity recovery of B-amylase produce by maltose fermentation. This effect must be due to complexation between B-amylase and maltrin resulting in a larger effective molecular weight. 54 3.6 LITERATURE CITED Higashihara, M. and S. Okada, "Studies on B-amylase of Bacillus megaterium strain no. 32", Agric Biol. Chem, 1974, 38:1023-1029. Hyun, H. H. and J. G. Zeikus, "General Biochemical Characterization of Thermostable Extracellular B-amylase from C. thennosulirrogenes", Appl. Environ. Microbiol., 1985, 49:1162-1167. Hyun, H. H., and J. G. Zeikus, " Regulation and Genetic Enhancement of Glucoamylase and Pullulanase Production in Clostridium thennohydrosulfuricum", J. Bacteriol., 1985b, 16421146- 1152. Lowry, O. H., N. J. Rosebrogh, A. L. Farr, and R. J. Randall, "Protein measurements with the Folin Phenol reagent", J. Biol. Chem, 1951, 193:265-275. Miller, G. L., "Use of dinitrosalicylic acid reagent for determination of reducing sugars," Anal. Chem, 1969, 31:426-28. Lamed, R. J., and J. G. Zeikus, "Novel NADP-linked alcohol-aldehyde/ketone oxidoreductase in thermophilic ethanologenic bacteria", Biochem. J., 1981, 195: 183-190. Robyt, J. F., and W. J. Whelan, "The [i-amylase", In J. A. Radley (ed.), Starch and its derivatives Chapman and Hall Ltd., London, 1968, 477-97. Saha, B.C., G-J. Shen and J.G.Zeikus, "Behavior of a novel thennostable B-amylase on raw starch", Enzyme Microb. Technol., 1987, 9:598-601. Shen, G-J, B. C. Saha, Y. E. Lee, L. Bhatnagar, and JG. Zeikus, " Purification and characterization of a novel thennostable B-amylase from Clostridium thermosulfurogenes", Biochem. J., 1988, 254. Schink, R., Y. Kurtimi and H. Nishira, "Production and Some Properties iof B-amylase of Baccillus sp. BQlO ", J. Ferment. Technol., 1975, 53:693-702. Schink, R. and J. G. Zeikus, "Characcterization of pectinolytic enzymes of Clostridium thermosulfurogenes", FEMS Letters, 1983, 17:295-298. Thomas, M. F., F. G. Priest and J. R. Stark, "Characterization of a Extracellular B-amylase from Bacillus megaterium sensu stricto", J. Gen. Microb., 1980, 118:67- 72. Takasaki, Y., "Purification and Enzymatic Properties of B-amylase and Pullulanase from Bacillus cerus var.", Agr. Biol. Chem, 1976, 40(8):1523-30. 55 Zeikus, J.G., "Thermophilic bacteria: ecology, physiology, and technology", Enzyme Microb. Technol., 1979, 1:243-52. CHAPTER 4 EFFECT OF FERMENTATION CARBON SOURCE ON THE PRECIPITATION OF B-AMYLASE FROM CLOSTRIDIUM THERMOSULFUROGENES 4.1. Abstract [S-amylase from Clostridium thermosulfurpgenes was recovered from cell free broth by ammonium sulfate and ethanol precipitation. The effects of the fermentation carbon source, temperature, and precipitant concentration on the B—amylase recovery process were studied and compared. Fifty percent saturated ammonium sulfate or twenty percent ethanol was the optimum concentrations for the enzyme recovery and purification. The B-amylase was found to form a soluble complex with starch dextrins in the fermentation broth. Light scattering studies revealed that the particle size of the precipitated complex was significantly larger for ethanol as compared to ammonium sulfate precipitation. Therefore, recovery of B-amylase activity was directly related to the particle size obtained. Smaller particles were obtained in the absence of starch; however, the ammonium sulfate resulted in larger particles as compared to the ethanol precipitation. The complexation of the B-amylase with the starch and subsequent precipitation of the complex can be interpreted as a bio-specific recovery process. The temperature effect on the precipitation process was also related to the carbon source 56 57 used in the fermentation. Gel filtration gave high B-amylase specific activity, and a single band or single peak was obtained with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or HPLC gel filtration for B-amylase produced with maltose. Two different molecular weight fractions were obtained for maltrin fermented B—amylase, and they were confirmed as two different single peaks in HPLC gel filtration. SDS-PAGE showed only a single band for both of these fractions in denatured form. These results further support the complexation of B-amylase subunits with starch dextrins, and the complex may be the reason for the higher effective molecular weight B-amylase observed. 4.2. Introduction Protein precipitation with the subsequent recovery of the precipitate is one of the most important unit operations in downstream processing. More than half of all purification schemes use a precipitation step somewhere in the process (Steven Oh, _e_t g, 1986). The potential applicability of precipitation is being expanded with the incorporation of novel precipitants and processes. The major role of protein precipitation as a separation process is in its ability to handle large quantities of dilute streams. This attribute allows precipitation to be used as an initial recovery process with the resulting product being further purified by more expensive techniques as the end use dictates. Although precipitation is used for both product recovery and purification, it is most effective in achieving the former. Precipitation is followed by a solid-liquid separation whose efficiency is dependent on the characteristics of the precipitate (Devenux pt .a_l., 1986). 58 4.2.2 Protein precipitation kinetics studies The understanding of protein precipitation requires the study of the formation of a stable solid phase, the subsequent aggregation, and efficient separation of the precipitate. In a destabilized protein solution a primary particle is formed by nucleation and it subsequently aggregates due to the diffusive motion of the particles. The final size of the particles is dependent on agglomeration and disruption. The primary particles continue to grow to a size where fluid dynamics become important and fluid driven particle-particle collisions lead to the formation and growth of final aggregates. These grth processes are termed perikinetic and orthokinetic aggregation according to zero and uniform shear fields, respectively (Bell 91 Q, 1983). However, in practice most precipitations are conducted in turbulent conditions, so other mechanisms and rate relations should be considered. Grabenbauer and Glatz (1981), in a study of isoelectric precipitation of soy proteins, have identified that the protein concentration, agitation, and ionic strength are all significant factors in shaping the particle size distribution. They found that protein concentration influences the number of aggregates more than it does their growth or break-up rates (Grabenbauer and Glatz, 1981). Agitation affects the growth rates of aggregates, but affects aggregates breakup much more. In other studies, protein precipitation is described as a rapid formation of submicron particles followed by aggregation via collision between particles. Collision efficiencies are high when one of the particles is small, but are quite low otherwise (Petenate and Glatz, 1983). On the other hand, particle size plays a major role in the recovery of precipitates. Because of similar densities of the protein precipitate and the aqueous phase, the 59 particle size of the precipitate is crucial to the effectiveness of particle removal (Petenate and Glatz, 1983). In fact, there is evidence that small particle size may be responsible for poor yields associated with some precipitation and recovery processes. Since centrifugal recovery is related to the square of the particle size, the improvement in the aggregate strength could result in a significant improvement in separation efficiency (Bell and Dunnill, 1982). The recovery of B-amylase from C. thermosulfurogenes produced at the Michigan Biotechnology Institute was studied using salting-out and organic solvent precipitation in conjunction with a biospecific interaction between B-amylase and starch dextrin molecules. The effects of fermentation carbon source, temperature, and protein concentration on precipitation of B—amylase recovery were studied. Crude B—amylase was further purified by a Bio-Gel gel filtration column and the different size fractions were collected. Higher B-amylase activity peak fractions were pooled and concentrated by UF (PM10, Plate membrane, 10,000 MWCO). The effective molecular weight and the purity of the enzyme were analyzed by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. Finally, the amino acid sequence of the B—amylase was determined and compared for each fermentation carbon source. 4.3 Materials and Methods 4.3.1 Production of fl-amylase. The thennostable, solvent resistant extracellular enzyme crude B-amylase was produced using trypticase and yeast extract (TY E) or corn steep liquor medium as the nitrogen source, and maltose, glucose, or maltodcxtrin (maltrin-10) as the carbon source. Two different Braun fermenters (l4 and 100 liters) and a 300 liter 60 Lab—Line-Bioengineering fermenter were used to produced B-amylase from Clostridium thermosulfurogenes. The fermentation broth was initially concentrated by ultrafiltration using a 30,000 MWCO spiral membrane (Amicon Spiral Ultrafiltration Cartridge model SlOY30). Subsequently, the cells were removed by centrifugation. The fermentation broth was purified about six fold during the above procedures. B-amylase specific activity of approximately 50 U/mg was used in all precipitation experiments. 4.3.2 Enzyme activity Ja‘nd protein assays Enzyme activity was determined by incubation of the enzyme solution with 2% soluble starch at 60°C, pH 6.0 for 30 minutes. The reducing sugars released by fl-amylase action on starch were estimated by the DNS (dinitrosalicylic acid) method. One unit of enzyme activity is defined as a micromole of maltose released per unit time under the conditions of the assay. Total protein concentration was estimated using the Lowry pt p1. (1951) assay method with bovine serum albumin as a standard. 4.3.3 Gel filtration A Bio-Gel column (1.5 cm x 150 cm) equilibrated with 50mM acetate buffer (pH 6.0) containing 50mM CaCl2 was used for gel filtration. Since, this was a small column, only 2-3 ml of concentrated B-amylase were purified at a time. The elution rate was 0.1 -0.2 ml/min. Fractions of B-amylase fermented with different carbon sources were collected and assayed for specific activity, total protein content, and total 61 carbohydrate content. The higher activity fractions were pooled, concentrated by UF (PM10, plate membrane, 10,000 mwco), and dialyzed against the same buffer. These concentrated peak fractions were used as purified enzymes for SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. 4.3.4 Molecular weight determination by HPLC gel filtration Purified enzyme fractions were chromatographed on a Protein Pak 3OOSW column (Waters Associates) equilibrated with 50mM acetate buffer, pH 6.0 with 5mM CaClz. A 100uM of enzyme sample was loaded and eluted with the same buffer at a rate of 1.0 ml/min. The protein peaks were detected by a UV detector and recorded by a Waters 700 recorder. The retention time for each B-amylase peak was obtained. Molecular weights and retention times were calibrated for different molecular weight protein standards. Effective molecular weights were estimated for B-amylase produced with maltose and maltrin. In order to compare the differences in B-amylase produced with maltose or maltrin, the amino acid sequence of electrophoretically pure li-amylase produced with maltose and maltrin were determined at the Department of Biochemistry, Michigan State University. 4.3.5 Polyacglamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis was performed for all gel filtered and concentrated enzymes as described by Laemmli (1970). Bovine serum albumin (66,000), egg albumin (45,000), glyceraldehyde 3-phosphate dehydrogenase (36,000), trypsinogen (24,000), carbonic anhydrous (20,000) and 62 a-lactalbumin (14,200) were used as standards. Gels were then stained with coomassie brilliant blue G-280 and compared. 4.3.6 S_ltirred batch precipitation studies Three different kinds of B—amylase protein mixtures were used in batch experiments. 1. B—amylase produced with maltose or glucose as the fermentation carbon source. 2. B—amylase produced with starch as the fermentation carbon source. 3. Synthetic B—amylase mixture: B—amylase precipitated by fifty percent ammonium sulfate was redissolved in acetate buffer (50mM), pH 6.0, containing 5mM calcium chloride and dialyzed against the same buffer for 24 hours. The above B-amylase was added to pretreated corn steep liquor and acetate buffer (50mM), pH 6.0, containing 5mM calcium chloride to match the protein concentration and B-amylase activity of the original fermentation broth. The results of preliminary batch precipitation studies indicated no significant difference in protein precipitation when TYE or corn steep liquor was used as fermentation medium nitrogen source or when maltose or glucose was used as fermentation carbon source. The optimum precipitant concentrations were 50% saturated ammonium sulfate or 20% (v/v) ethanol for stirred batch precipitations of B-amylase. These concentrations were used in all subsequent stirred batch precipitation studies. 63 Concentrated B-amylase fermentation broth and the synthetic B-amylase mixtures were used for stirred batch precipitation studies. Saturated ammonium sulfate or ethanol was added dropwise to 75ml of [i-amylase solution with gentle stirring (using a rubber coated stirring rod) in a 250 ml beaker. The final concentration of the mixture was 50% saturated ammonium sulfate or 20% (v/v) ethanol. Each batch was stirred for two hours at a stirring rate of 80 rpm. The precipitate was collected by centrifugation (12000 rpm: 30 min: 4°C), redissolved in 20 ml acetate buffer (50mM), pH 6.0, containing 5mM calcium chloride, and dialyzed against the same buffer for 24 hours. Protein assay and activity tests were performed before and after the precipitation. 4.3.7 Protein precipitation kinetics Ltudies A Coulter Electronics'Model N4 sub-micron particle analyzer was used to measure the particle size distributions of the protein precipitate. An auto-sampling time of 200 seconds was used in all experiments. The auto-correlation functions were generated and transformed into particle size distribution through standard estimation techniques, yielding the mean size and standard deviation. B-amylase with the precipitant were mixed in a continuous stirred batch reactor and 2m] samples were analyzed at different time intervals. A Perldn-Elmer Lambda 3A spectrophotometer was used to measure the turbidity at 660 nm (i.e, absorbance) of the protein precipitate with time. The kinetics of initial particle formation were monitored immediately after mixing using 50% saturated ammonium sulfate or 20% ethanol to B-amylase broth produced with maltose or (,4 maltrin. 50% saturated ammonium sulfate or 20% ethanol solution was used as blank for the above experiments. In addition, 1% soluble starch was precipitated with 50% ammonium sulfate or 20% ethanol and initial kinetics studies were also performed and compared. 4.4 Results 4.4.1 _B_atch precipitation stadies Preliminary batch precipitation results for different concentrations of ammonium sulfate and ethanol are reported in tables 4.1 and 4.2, reSpectively. The temperature and the carbon source effect on total protein and B—amylase activity recovery with 50% saturated ammonium sulfate are shown‘ in Figures 4.1 and 4.2, respectively. Total protein and activity recoveries were generally better for B-amylase with maltose than than with maltrin as the carbon source. However, at 50°C B-amylase recovery dropped for B—amylase produced with maltose. The effects of temperature on total protein and B—amylase activity recovery for ethanol precipitate are shown in Figures 4.3 and 4.4, respectively. The maximum specific activity recovery occurred at room temperature. Further increases in temperature resulted in precipitation of other proteins without additional precipitation of B—amylase. The activity recovery was enhanced by the strong binding of B—amylase to starch dextrins and the resulting low solubility of the complex in aqueous ethanol. The addition of maltrin-10 to the maltose containing broth resulted in a 3 fold increase in activity recovery with ethanol precipitation and is shown in Table 4.3. 65- TABLE 4.1 Purification of B—amylase by different concentration of ammonium sulfate precipitation (carbon source: maltose). steps Specific Purification Activity activity ratio recovery (U/mg of protein) (%) Pretreated 26.0 1 100 broth 50% 174.0 7 98 ammonium sulfate ppt. 50-65% 4.0 - 1.4 ammonium sulfate ppt. Experiments were conducted at room temperature for two hours stirring. Purification ratio = (final specific activity/ initial specific activity) 66 TABLE 4.2 Purification of B-amylase by different concentration of ethanol precipitation (Carbon source: maltrin) Steps Specific Purification Activity activity ratio recovery (U/mg of protein) (%) Pretreated 57.5 1 100 broth 20% ethanol 358.7 6.2 72 precipitation 30% ethanol 241.6 4.2 87 precipitation 40% ethanol 120.9 2.1 74 Experiments were conducted at room temperature for two hours stirring. Purification ratio = (final specific activity / initial specific activity). Protein Recovery (%) 67 I 00. jF—T-T‘tfi‘T‘flhl 1 1—r 1 1 1 I 1 TiT 1 I 1 1 rfi I r I 1 r .r O 1 ’,o~~_p~ 4 9 .j ’,.””’ ~~~~~ 0 j .4 ,,,,,,, o” - cl 0- flflflflfl .1 80 - - " '1 700‘ —‘ .1 I 60.— -— 50.A .— .. _____ .3 .. T '0‘ “““““ "‘ .4 *_~ ’,”” .. a ~~~~~~~~ Vf” ., 400— _ - .l d d . sen-at starch .. 30 ‘ e-o glucose ‘ . TT—T r [*1 r r 1 I r 1 rfi r 1 1 r r I 1 1 j 1 I 1 1 1 T Temperature (QC) Figpre 4.1. Effects of temperature and carbon source on total protein recovery of B—amylase precipitation with 50% saturated ammonium sulfate 0. 10. 20. 30. 40. 50. 60. ' Activity Recovery (70) " its-3e starch e-o glucose 500 I I I I' I I I I I I I I I I l I I I I I I I I I I T I I O. 10. 20. 30. 40. 50. Temperature (CC) Figure 4.2. Effects of temperature and carbon source on B-amylase activity recovery during B-amylase precipitation with 50% saturated ammonium sulfate I 60. Protein Recovery (73) 501—1—‘1 1‘! “I 1—r 1 1 1 rfir 1 1 I 1 1 1 ‘1 I r 1 r 1 I TI 1 1 n 40.-— — .J - .l - .J .. Q or \ c- \\ 30.— .— - \\\ III/0 : " \\ /’ \ x’ .. "‘ \\ [’1' .1 \\\ {35.71 __________ g .. 20. -— III-Nu D._._—::;, ._ .1 ~~~~~ *2” .1 .1 .1 10.-— .J ‘ ill—BIS Starch ‘ ‘ o-e glucose " 0. 1 r r r I 1 1 FT I 'T— r 1 1 I FT fir 1—1 1 1 1 I 1 1 I T O. 10. 20. 30. 40. 50. 69 Temperature (00) Figure 4.3. Effects of temperature and carbon source on total protein recovery of B-amylase precipitation with 20% (v/v) ethanol 60. Activity Recovery (73) 100. 90. 80. 70. 60. an .0 40. 30. 20. 10. Figure 4.4. Effects of temperature and carbon source on B—amylase activity recovery during B—amylase precipitation with 20% (v/v) ethanol 70 q 1 _ 1 1 r I 1 1 1 r I 1 1 I rj 1 r T—F‘j 1 r 1 r I 1 r r -I -4 .. A- Z _, I \ .—4 .1 I \\ or c- I \\\ C1 .. I \\ - \ “ l _ \ .. II \\\ I .. ll \\ .1 .: l \\ .— [I \\ .. - I 7 ' .: x 3 - .l '2 ”I .. ax .. ~ \ - a: \\ '1 q \\ o-‘T’"o : .. \\ ’av"’ .. .. \ .9” \ ______ .. on: O—‘_-.- .— - d .. .2 . are—are starch I q . G—o glucose 1 r r r I r 1 r I r 1 r 1 I r r 1 1 I 1 r 1 1 I r r I O. 10. 20. 30. 40. 50. Temperature (0C) 60. 71 TABLE 4.3 Effect of starch dextrin on B-amylase recovery with 20% ethanol. B—amylase was initially concentrated by ultrafiltration. Starch dextrin Total activity conc. recovery (%) (%) Concentrated 0.0 15 B-amylase Concentrated 0.5 50 B-amylase Experiments were conducted at room temperature for two hours stirring. 72 4.4.2 Fundamental kinetic sttulies The change in mean particle size with time after mixing for two different precipitants and fermentation carbon sources is shown in Figure 4.5. Light scattering studies showed that the particle size of the precipitated complex was significantly larger when ethanol was used as precipitating agent in presence of starch B-limit dextrins. Mean particle size of the precipated complex increased with time up to two hours, and decreased afterwards for ethanol when starch B-limit dextrins were present in the fermentation broth. Particles were initially formed and maintained a constant mean size with time for the other three conditions. Ammonium sulfate precipitate had a larger mean particle size than ethanol in the absence of starch dextrins. Pure soluble starch dextrins (1%) was precipitated with 50% ammonium sulfate or 20% ethanol to compare precipitate growth (if any) in the absence of fi-amylase. When soluble starch was precipitated with ethanol, particles were formed quickly and increased in size rapidly with time but no precipitate was obtained with ammonium sulfate. Mean particle size reached the instrument’s measuring limit within few minutes for ethanol. This indicates that the B—limit dextrins are precipitated by ethanol. Change in relative absorbance (turbidity) with time is shown in Figure 4.6. Figure 4.6A shows that particles are larger for ethanol precipitation when starch is present and increase with time slowly to a maximum size. A decrease in mean particle size occurred in few hours after mixing. A sharp increase in absorbance was observed (Fig. 4.63) immediately after mixing for starch dextrins fermented B-amylase with ammonium sulfate with similar results for the other treatments. Mean Particle Size (mm) 73 12000 ' ‘I I I I ‘ T I U l I ‘l 1' I ‘ Ar-A 1% maltose. 20% ethanol : rte-x 1% starch. 50% ammonium sulfate . -‘ 9-0 1% maltose. 50% ammonium sulfate i 1000": H 1% starch. 207.? ethanol ' . l ' - 800 - - ‘ 1 600.-— .1 3 e 400.— 200.— O I I 1 I I I I I fit I I I I O 100. 200 300. Time (min) Figure 4.5. Change in mean particle size with time for B-amylase precipitate with 50% saturated ammonium sulfate and 20% (v/v) ethanol B-amylase was produced by fermentating maltose or maltodcxtrin and was initially concentrated by ultrafiltration 74 l l l l l l D .l .i A " °‘ C v Q - - (D (D an C" .4...) U a) ‘ ' O C O .0 L. .. u 0 (I) .0 .4 B- O Q) _ .. .2. .44 .9. (D 0: .. - _A . .l 111111111111111111111111]1111rr111111111111111111 010 20 3O 4O 50 60 7O 80 90100 Time after mixing (min) Figure 4.6. Change in relative absorbance (turbidity) with time for B-amylase precipitate with 50% saturated ammonium sulfate and 20%(v/v) ethanol. B—amylase was produced by fermentating maltose or starch dextrins and was initially concentrated by ultrafiltration A» Maltose-ammonium sulfate, B- Maltose-ethanol Cu Maltodextrin-ammonium sulfate D-- Maltodextrinuethanol 75 Photomicroscopic pictures were taken at two different time intervals for B~amylase precipitates with ethanol. The pictures are shown in figure 4.7A and B. The breakage of protein clusters due to longer stirring is obvious in figure 4.78. 4.4.3 Molecular weight estimation The protein contaminants were removed by gel filtration on a Bio-Gel column. Total protein content and B—amylase activity profiles for B-amylase produced with maltose is shown in Figure 4.8. Profiles for B—amylase produced with maltrin are shown in Figure 4.9. Only one B—amylase activity fraction was obtained for B-amylase produced with maltose but two different activity fractions were obtained for B-amylase produced with maltrin. The final yield and purification for B‘amylase are shown in Table 4.4. SDS-polyacrylamide gel electrophoresis in denatured form showed a single band for B-amylase produced with both carbon sources (Figures 4.10 and 4.11). Likewise, the HPLC gel filtration column gave single peak for higher B-amylase activity fractions and are shown in Figures 4.12 to 4.14. The gel permeation purified enzyme was considered to be homogeneous by detection of a single peak on SDS-PAGE and a single peak on HPLC gel filtration column. Although the first and second maximal activity fractions from B-amylase produced with maltrin showed single peaks for HPLC gel filtration, their different retention times show that they have different effective molecular weight or size. An estimation of total carbohydrate content didn’t reveal any correlation among the fractions. Carbohydrate contents are similar and distributed among all fractions. The amino acid sequences for B-amylase produced 76 .r . . '33.? ”.4 ‘ ~ "2 ' was, 5 W;,._ a” - a: '. A“! Mb" .4“ ,\ i ..W h .. . ” ... .9, an a. ... fl, ....» :23 .. a... ..., aw» .\_ tes alter minu broth during tures taken at 90 and 150 re the addition of ethanol to B—amylase fermentation ic p' rcroscop Photom Figure 4.7. res tud' 'tation s precrpr 77 TABLE 4.4 Major steps for purification of B-amylase from C. thermosulfurogenes Steps Total Total Purification yield protein activity fold (%) (mg/ml) (U) Original Broth 6534 69428 1.0 100 Ultrafiltration 730 55266 7.6 80 Precipitation 28 52166 179.0 75 Gel filtration 4 17765 435.0 . 26 Fermentation Carbon Source : 1% maltose Membrane Size: 30,000 MWCO Cartridge Membrane Type: Diaflo Hollow Fiber (Amicon) Final B—amylase activity: 4562 U/mg 78 200.0 2.0 I H Activity : G—O O-D. >- 175.0: A \ r or .0 o 1 (Ll/ml) 100.0 Activity 3 O tn .0 o 25.0 llllllIllllllilllllllllllllll 0.0 Fraction Number Fiou, re 4.8. Activity and total protein distribution for B-amylase through a Bio-Gel column. Carbon source was maltose and B-amylase was initially concentrated by ultrafiltration and ammonium sulfate precipitation 79 125.0 5.0 . x—x Activity ‘ 0—0 0.0. I 53 - l00.0- i L40 . i l- .i l b- A l __ . r l- E . l . \75 0— ; ~10 3 ~ 1 L >\ d i b :4: - l- ..>.. ' “ «Sisao— ‘2-0 (l q r h - D ‘ _ .4 t 1 t- 25.0-4 ° -1.o .. 0‘06 l- : 0. o” q k .40“, " g:c:,aadnc3dflcg “caanc:=°¢c:a- L: 0.0 ”3.55:." ‘a--.-.=:e-=:=':'§l°--» i—0.0 0 5 10 15 20 25 30 35 40 45. _50 55 60 Fraction Number Figure 4.9. Activity and total protein distribution for B-amylase through a Bio-Gel column. Carbon source was maltodextrin and B-amylase was initially concentrated by ultrafiltration and ethanol precipitation 80 1‘?“ ‘T o t ‘ ‘ 7 ' “-»-=.;,.-,~:... l e 1 FIGURE 4.10 SDS-polyacrylamide gel electrophoresis of purified B-amylase. Carbon source was maltose. The B-amylase was electrOphoresed on 10% acrylamide gel and stained with Coomassie Brilliant blue R-ZSO. B— bio-gel filtration or _(_I- combination of 3% ammonium sulfate and 20% ethanol; 5; and _Q- standard marker proteins. The standards were: bovine serum albumin (66,000); egg albumin (45,000); glyceraldehyde 3-phosplrate dehydrogenase (36,000); trypsinogen (24,000); carbonic anhydrous (20,000); and or-lactalbumin (14,000). 81 ll illll FIGURE 4.11 SDS-polyacrylamide gel electrophoresis of purified B—amylase. Carbon source was maltodcxtrin. The B-amylase was electrophoresed on 10% acrylamide gel and stained with Coomassie Brilliant blue R-250. _B- bio-gel filtration, _A_- standard marker proteins. The standards were: bovine serum albumin (66,000); egg albumin (45,000); glyceraldehyde 3—phosphate dehydrogenase (36,000); trypsinogen (24,000); carbonic anhydrous (20,000); and a-lactalbumin (14,000). Absorbance at 280 nlvl 82 53 '52 :2 o «C. «5 Time (Min) FIGURE 4.12 Separation of gel pure B—amylase (previously separated by Bio-Gel column chromatography. The dialyzed B—amylase was applied to a column (l.5cm*150cm) equilibrated with 50mM acetate buffer pH 6.0 with CaCl1 and eluted with same buffer. 5ml fraction were collected and B- amylase specific activity and total protein content were assayed.) by a HPLC protein pak 3005w gel filtration column. Carbon source was maltose. 20$; Absorbance at 280 nlvl 83 000’ f 3 l5“ 7359‘ . ll 25 IS 00 Time (Min) FIGURE 4.13 Separation of gel pure B-amylase (previously separated by Bio-Gel column chromatography. The dialyzed B-amylase was applied to a column (lScm‘lSOcm) equilibrated with 50mM acetate buffer pH 6.0 with CaCl2 and eluted with same buffer. 5ml fraction were collected and B- amylase specific activity and total protein content were assayed.) by a HPLC protein pak 3005w gel filtration column. Carbon source was maltodcxtrin. First peak fraction by gel filtration. Absorbonce at 280 nlvl 84 W I m (x. m Time (Min) FIGURE 4.14 Separation of gel pure B-amylase (previously separated by Bio-Gel column chromatography. The dialyzed [S—amylase was applied to a column (1.5cm*150cm) equilibrated with 50mM acetate buffer pH 6.0 with CaCl, and eluted with same buffer. 5ml fraction were collected and [3- amylase specific activity and total protein content were myed.) by 3 HPLC protein pak 300$w gel filtration column. Carbon source was maltodcxtrin. Second peak fraction by gel filtration. 85 from C.thermosulfurogenes with maltose and maltrin and from Bpolymga are shown bellow. l 5 10 15 I Ser-Ile-Ala-Pro-Asn-Phe-Lys-Val-Phe—Val-Met-Gly-Pro-Leu-Glu- II -lle-Ala- -Asn-Phe-Lys-Val-Phe- -Met-GIy-Pro-Leu-Glu- III . Ala-Val-Ala-Asp-AspoPhe-Gln-Ala-Ser-Val-Met-Gly-Pro-Leu-Ala- I 0amylase produced with maltose II [ii-amylase produced with maltrin III B—amylase from Bpolymyxa These results show that B-amylase produced from C.thermosulfurogenes is different from that produced from Bpolymfla thereby showing the sensitivity of the sequence. The use of different carbon sources didn’t reveal any significant difference in sequence. 4.5 Discussion In the preliminary experiments, the B—amylase activity and the assay results for 40% saturated ammonium sulfate precipitation showed that a considerable amount of B«amylase activity was left in the supernatant. The 50% saturated ammonium sulfate precipitation gave the best recovery and purification (T able 4.1). The supernatant of 86 the 50% precipitation was reprecipitated with 65% of the ammonium sulfate resulting in a precipitate with a very low activity. For ethanol precipitation 30% WW) ethanol showed higher recovery than for 20%, but the purification ratio was low due to the precipitation of unwanted proteins (Table 4.2). Therefore, it was concluded that 50% ammonium sulfate or 20% (v/v) ethanol were the optimum conditions for recovery and purification ratio. It was also found that there was no significant difference in protein recovery when the fermentation medium was TYE or corn steep liquor. Furthermore, no difference precipitate formation or activity recovery was found between B-amylase produced with maltose or glucose; however, the results were different from TYE or corn steep liquor. Therefore, maltose and maltrin-10 were used for the protein recovery, and activity experiments, and in fundamental kinetic studies. Initially, fermentation broth was concentrated by spiral membrane ultrafiltration. The choice of UF with a high molecular cut off spiral cartridge resulted in relatively high fluxes. At the same time a rather large portion of small peptide proteins was removed with the filtrate. Therefore, protein specific activity was increased more than six fold during UP and centrifugation. The effect of temperature and fermentation carbon source on total protein and activity recovery could be related to the kinetics of nucleation/agglomeration and solubilities of proteins in the broth. This effect could be explained very clearly by first explaining the particle formation and agglomeration behavior given in Figures 4.5 and 4.6. The change in mean particle size and relative absorbance (turbidity) follow similar behavior with time for corresponding precipitation curves (Figures 4.5 and 4.6). Since the light scattering instrument requires 200 seconds of sampling time it was 87 impossible to obtain initial particle formation (nucleation) kinetics, but these were studied by turbidity measurements (Fig. 4.6). Similar turbidity curves were obtained by Chan 33 _a_l. (1986) in soy protein precipitation with ammonium sulfate and ethanol. Particle size is highly dependent on protein concentration and the concentration of precipitants. The primary particles aggregated by further collision, the final size being controlled by a balance between growth and break up, which are both shear controlled. Break up is dependent on concentration (Virkar gt £11., 1982 and Twineham e_t §l_., 1984), on the shear history of the particles (Bell and Dunnill, 1982), and on the degree of binding between the basic particles. In ethanol, starch dextrins cause particles to aggregate with mixing for about two hours and break up with further mixing. This phenomenon is further supported by the photomicroscopic pictures taken at 90 and 150 minutes after the ethanol addition to B-amylase solution. Figure 4.7A shows the agglomeration of the protein precipitates and 478 shows the breaking up of the aggregates at longer stirring time. This is why longer times of stirring resulted in less protein activity recovery for ethanol precipitation. Several mechanisms have been proposed to describe the break up of the precipitate particles (Petante and Glatz, 1982), although a satisfactory theoretical prediction is not available. The mean particle size of the ammonium sulfate precipitate from B—amylase produced with maltose was greater than the size of the precipitate from li-amylase produced with maltrin and resulted in better protein and activity recovery for maltose fermented protein (Figures 4.1 and 4.2). An increase in solubility of the precipitate at 50°C for ammonium sulfate may have resulted in less protein recovery in Fig. 4.1. The salting-out process was inhibited by the presence of starch dextrins and B—amylase 88 was readily recovered at ambient temperature with 50% saturated ammonium sulfate from B—amylase produced with maltose. The ethanol precipitated B-amylase produced with maltose had a smaller mean particle size (Figure 4.5) and relative absorbance (Figure 4.6) as compared to Bamylase produced with maltrin. A 1% soluble starch ethanol precipitate tended to aggregate very fast and reached 10mm in a few minutes. Comparing these three levels of particle size or relative absorbance (turbidity) showed that the formation of the soluble complex with starch dextrins in the fermentation broth inhibited the starch precipitation and/or enhanced the protein activity recovery. The activity recovery was enhanced by the strong binding of B-amylase to starch dextrins and the resulting low solubility of the complex in aqueous ethanol. The large difference in activity recovery for starch fermentation (Figure 4.4) and no significant difference in total protein recovery (Frgure 4.3) support the notion that enzyme-starch dextrins adsorption is a biospecific adsorption. Microbial B-amylase is known to be adsorbed on starch (Hoshino e_t gl_., 1975). The addition of soluble starch to the glucose containing broth resulted in a 3 fold increase in activity recovery with ethanol as a precipitant (Table 4.3). This result confirmed the previous observations and indicated that ethanol precipitation of B-amylase in the presence of starch dextrins is a biospecific precipitation. The major protein contaminants were separated by gel filtration. Pure B—amylase was obtained for B—amylase produced with maltose and two pure fractions were obtained from B-amylase produced with maltrin. B-amylase produced with maltose and the second fraction of B—amylase produced with maltrin show the same molecular 89 weights, which is about 60,000 daltons determined by HPLC gel filtration column and about 50,000 daltons in denatured form determined by SDS-PAGE. The first fraction obtained in B—amylase produced with maltrin shows about 228,000 daltons determined by HPLC gel filtration; but, the same molecular weight was obtained in the denatured form by SDS-PAGE. The difference in effective molecular weights could be explained by three different hypotheses: 1. B—amylase subunits (each 60,000 daltons) form nonstable clusters among themselves and the clusters are stabilized by the presence of dextrins, but they are separated by sodium dodecyl sulfate. 2. The dextrins present in the fermentation broth form a biospecific complex with different amounts of B-amylase subunits resulting in higher effective molecular weight complex. They are also breakable in denatured form. In a different study, 85% of B—amylase activity was adsorbed by raw starch from crude broth for B-amylase produced with maltose but very little was adsorbed for B-amylase produced with maltrin. This experiment supports that the starch dextrins binding sites are already bound with maltrin in 0amy1a5e produced with maltrin. This phenomenon is called substrate-enzyme complexation. 3. B—amylase produced with maltrin is entirely different from that produced by maltose. Both are produced only by the micro-organism C.thermosulfurogenes. The amino acid sequencing study showed that there is no difference between both carbon sources produced B—amylase. To narrow down these hypotheses further enzymological studies and binding studies are required, and they are beyond the scope of this project. 90 In summary, the above results show that the B-amylase downstream purification is dependent on the carbon source (substrate) used in fermentation. Maltose or glucose fermented B-amylase results in smaller mean particle size and can be precipitated by 50% saturated ammonium sulfate. On the other hand, B-amylase produced with starch dextrins shows a larger mean particle size and is easily precipitated by 20% ethanol. Only three precipitation steps were needed to purify Boamylase to electrophoretically homogeneity. The specific activity of B-amylase purified from C.thermosulfurogenes was high (~4,000 U/mg) when compared to the enzymes from Thermogctinomyces (408 U/mg) by Obi and Odibo(1984) or from sweet potato (560 U/mg) (Bemfeld, 1955). 4.6 Conclusions 1. The mechanism of the precipitation of B-amylase with ammonium sulfate and ethanol depends on the fermentation carbon source. Fifty percent saturated ammonium sulfate or twenty volume percent ethanol is the optimum concentration for the recovery and purification of C.thermosulfurogenes derived B-amylase for salting out or organic solvent precipitation, respectively. Room temperature is the optimum temperature for B-amylase recovery and purification with both agents. Ammonium sulfate precipitation results in higher activity yield from fermentation broths with maltose as a carbon source. Starch dextrins present in the fermentation broth form a biospecific 91 complex with B-amylase which could be easily precipitated with ethanol. . Ethanol precipitation of B-amylase gives higher recoveries with starch dextrin containing broths and is a biospecific recovery process. . Three purification steps are enough to purify the B-amylase to homogeneity with B-amylase specific activity about 4,000 U/mg. . B—amylase produced with starch dextrins has higher effective molecular weight (228,000 daltons) clusters formed by B—amylase subunits. . About 50,000 daltons molecular weight was obtained in denatured form with maltose and maltrin determined by sodium dodcyl sulfate polyacrylamide slab gel electrophoresis. 92 Literature cited Baily, J. E. and D. F. Ollis. Biochemiczflingineering Fundamentals. 2nd Ed., McGraw-I-lill, New York, 1986. Bell, D. H., M. Hoare and P. Dunnill. "The formation of protein precipitates and their centrifugal recovery", Adv. Biochem. Eng/Biotechnol., 1983, 26:1-72. Bell, D. J. and P. Dunnill, "The influence of precipitation reactor configuration on the centrifugal recovery of isoelectric soya protein precipitate", Biotechnol. Bioeng., 1982, 24, 11:2319-36. Bemfeld, P., "Amylases or and [3", Methods of Enzymol., 1955, 1:149-58. Chan, M. Y. V., M. Hoare and P. Dunnill. "The kinetics of protein precipitation by different reagents", Biotechnol. and Bioeng., 1983, 28:387-93. Devenux, N. and N. Hoare. "Membrane separation of protein precipitates: Studies with cross flow in hollow fibers", Biotechnol. Bioeng., 1986, 28:422. Elankovan, P. and K. A. Berglund. "Bemylase recovery from fermentation broths" Short term progress report, Michigan Biotechnolgy Institute/Michigan State University Traineeship, 1986. Flygare, S., T. Griffin, P. Larsson, and K. Mosbach, ”Affinity precipitation of dehydrogenases", Anal. Biochem., 1983, 133, 2:409-16. Grabenbauer, G. C. and C. E. Glatz, "Protein precipitation - analysis of particle size distribution and kinetics", Chem. Eng. Commun., 1981, 12:203-19. Hoshino, M., Y. Hirove, K. Sano, and K. Mitougi, "Adsorption of microbial B—amylase on starch", Agr. Biol. Chem., 1975, 39, 12:2415-16. Lowry, O. 11., N. J. Rosebrogh, A. L. Farr, and R. J. Randall, "Protein measurements with the Folin Phenol reagent", J. Biol. Chem, 1951, 193:265-75. Laemmli, U. K, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (London), 1970, 227:680—85. Nelson, C. D. and C. E. Glatz, "Primary particle formation in protein precipitation", Biotechnol. Bioeng., 1985, 27, 10:1434-44. 93 Petenate, A. M. and C. E. Glatz, "Isoelectric precipitation of soy protein. 1: Factors Affecting particle size distribution", Biotechnol. Bioeng., 1983, 25:3049. Oh, S. J. B., M. H. Hoare and P. Dunmill. "Protein purification", Bio/Technology, 1986, 4:954—8. Twineham, M., M. Hoare, and D. J. Bell, "The effects of protein concentration on the break-up of protein precipitate by exposure to shear", Chem. Eng. Sci., 1984 39, 3:509-13. Virkar, P. D., M. Hoare, M. Y. Y. Chan, and P. Dunnill, "Kinetics of the acid precipitation of soy protein in a continuous-flow tubular reactor", Biotechnol. Bioeng., 1982, 24, 4:871-87. CHAPTER 5 IMPROVED RECOVERY AND HIGH PURIFICATION OF eAMYLASE FROM CLOSTRIDIUM THERMOSULFUROGENES BY COMBINATION OF ORGANIC SOLVENT AND SALT PRECIPITATION 5.1 ABSTRACT An thermostable, extracellular, solvent resistant B-amylase from _C_ thermosulfurogenes was purified to 205 fold with 72 percent of total activity recovery by the combination of ethanol and ammonium sulfate precipitation in a batch reactor. The purification fold and the total recovery were highly dependent on the ethanol and ammonium sulfate concentration. The protein precipitates were stable and tend to aggregate with time. This altemative technique is a simple, low cost, short time process compared to other traditional purification methods. 5.2 INTRODUCTION Major development in genetic engineering and biotechnology has successfully produced a wide spectrum of protein products for food and health industries. These new proteins are produced either by large-scale bacterial fermentation or by mammalian cell culture. The large scale fermentation of these products has increased 94 95 the need for an efficient downstream protein concentration and purification scheme. In spite of the increasing need for large-scale purification, there is very little information available on separation techniques which are more efficient to replace the traditional methods. Precipitation of protein by a variety of reagents is an important stage in their recovery and fractionation (Bell 9; _a_l_., 1983) and more than half of purification schemes use a precipitation step, mostly with ammonium sulfate (Bonnerjea, 1986). Ammonium sulfate is one of the best known neutral salts used for precipitation by salting-out and ethanol is the most widely used agent for bringing about precipitation by reduction of the dielectric constant of the medium. Ammonium sulfate presents costly disposal problems in large scale applications, but organic solvents may be used on a large scale since they can be recycled efficiently. On the other hand the tendency for alcohol-water mixtures to denature protein is also well established (Schubert and Finn, 1981). There are advantages to the combination of salt and solvent precipitation. In organic solvent precipitation, salt itself is not the precipitating agent. The addition of small amounts of solvent and salt have opposite effects on the solubility of protein. The neutral salts, in general, increase the solubility of proteins in the presence of organic solvents. The balance between the precipitating action of organic solvents and the interaction with salts permits attainment of a variety of conditions under which the protein to be separated may be brought to any desired solubility (Cohn e_t. g, 1946). This balance is different at constant ethanol and salt concentration for each pH, temperature, and each protein components. This paper will focus on the 96 combination of two traditional protein precipitation techniques, organic solvent and salt precipitation to obtain highly purified B-amylase from C. thermosulfurogenes. B—amylase is an exo-splitting enzyme which attacks amylase, amylopectin, and glycogen from the nonreducing terminal, resulting in the formation of maltose in the B—configuration. B—amylase is specific for (It-1,4 linkages and is unable to bypass a-1,6 branch points. It is a commercially important enzyme useful for food and beverage industries. B—arnylase is widely distributed in plants and also has recently been found in the microbial world. Hyun and Zeikus(1985) have developed and characterized a novel thennostable extracellular B—amylase from Clostridium thermosulfurogenes, that is stable and optimally active at 75°C (Hyun and Zeikus, 1985). Some microbial B—amylases are reported to adsorb onto raw starch (Hoshino e_t. §_l_., 1975) and digest it (Ueda and Marchal, 1980). The specific binding of an enzyme to its substrate has been extensively studied in several cases to constitute a useful purification step (Wilchek 9;. §_l_., 1984). The raw starch adsorption, elution and digestion behavior of the B—amylase from _C_. thermosulfurogenes were studied and reported by Saha e_t. a (1987). The present study will introduce an alternative method to purify [iamylase by combination of ethanol and ammonium sulfate precipitation. 5.3 MATERIAL AND METHODS Cultivation of organism and concentration of Eamylase: The catabolic repression resistant mutant H-12-1 derived from Q thermosulfurogenes wild strain 4B (ATCC 33743) was used (Hyun and Zeikus, 1985). 97 The culture was grown anaerobically in a 300 liter fermenter containing 250 liters TYE medium with 1.0% maltose as carbon source (substrate) at 60°C for 10 hours. The culture broth containing cells was initially concentrated 8 times by a 30,000 MWCO spiral ultrafiltration membrane (model DCIO, Amicon) and the cells were separated by centrifugation (10,000 rpm, 30 min). Precipitation studies were conducted in a stirred batch reactor. First, ethanol was added dropwise to the concentrated B-amylase and then ammonium sulfate was added while stirring. Each experiment was stirred for two hours at room temperature. The precipitate was collected by centrifugation (18,000 rpm, 30 min), dissolved in acetate buffer (50 mm, pH 6.0 with 5 mm CaCl,_), and dialyzed against the same buffer. B—amylase activity and total protein were estimated before and after each precipitation study. The purity of the B-amylase was analyzed with a high pressure liquid chromatography (HPLC) protein pak 300 SW gel filtration column and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (Laemmli, 1970). The purity of the enzyme was compared with Bio-Gel filtered pure B-amylase. The change in mean particle size with time was measured with a Coulter N4 submicron particle analyzer. Two different particle _ kinetic studies, 1) 20% (WW ethanol with 5% ammonium sulfate, 2) 30% ammonium sulfate with no ethanol, were conducted and compared. Engyme assays: B—amylase activity was assayed using a 5ml reaction mixture consisting of 2% boiled soluble starch solution, acetate buffer (50 mm, pH 6.0 with 5 mm CaCh) and appropriately diluted enzyme solution. After incubation for 30 minutes at 60°C, the reducing sugar released was measured by the dinitrosalicylic acid method (Miller, 98 1969). One unit of B—amylase activity is defined as the amount of enzyme that releases 1 mM mole of reducing sugar as maltose per minute under the above conditions. Total protein was estimated by the method of Lowry 9;. £11., with bovine serum albumin as standard (Lowry e_t. a_l., 1951). 5.4 RESULTS Table 5.1 summarizes B-amylase specific activity, total activity recovery, and one step purification fold for different concentrations of ethanol and ammonium sulfate. 20% ethanol with 3% ammonium sulfate precipitation gave about seventy-eight percent B-amylase activity recovery with 31 times purification in a single purification step. Purity of B-amylase for the above combination was analyzed by gel filtration and the results are shown in figure 5.1. B—amylase purified by gel filtratiOn is shown in figure 5.2. Comparing these two figures only a small amount of impurity is present in the combination of precipitation. For the same experiment SDS-PAGE also shows a small amount of impurity (figure 5.3). The effect of agglomeration/aggregation after addition of a combination of precipitants and precipitation only by ammonium sulfate, are compared in figures 5.4. Particles were stable and tended to grow or aggregate with time for the combination of precipitation (figure 5.4) but particles were formed immediately after mixing and maintained their size throughout for ammonium sulfate ‘ precipitated B—amylase. Specific activity and recovery results for each purification step involved in B—amylase partial purification is given Table 5.2. 99 TABLE 5.1 Precipitation of B—amylase with combinations of ethanol and ammonium sulfate. The activity recovery and purification fold for different concentration of ethanol and ammonium sulfate precipitate. Ethanol Ammonium Specific Purifi- Activity conc. Sulfate Activity cation Recovery (%) (%) (U/mg) ratio (%) 0 30 2.37 8.3 93 20 O 125 4.4 23 10 5 298 10.4 11 10 10 322 l 1.3 22 10 20 785 27.5 88 20 3 889 3 1.0 78 20 5 867 29.7 59 20 10 661 23.2 40 20 20 442 15.4 60 30 10 25.3 -- 5 30 20 14.2 -- 3 B—amylase with cells was initially concentrated 8 times by 30,000 MWCO spiral UF membranes and the cells were separated by centrifugation (10,000 rpm, 30 min) prier to precipitation. Absorbance at 280 nM 100 0 80 3.75' . 7,.59‘ ll 25 IS 00“ Time (Min.) FIGURE 5.1 Separation of B—amylase (previously precipitated with 3% ammonium sulfate and 20% of ethanol) by a HPLC protein pak 300m gel filtration column. Area under the smaller peak shows the amount of impurity left with B—amylase. Absorbonce at 280 MA 101 2 83 '8 Time (Min.) FIGURE 5.; Separation of gel pure B-amylase (previously separated Bio-Gel column chromatography of B-amylase. The dialyzed enzyme was applied to a column (1.5cm’150cm) equilibrated with 50mM acetate buffer pH 6.0 with CaCl2 and eluted with same buffer. 5ml fraction were collected and B-amylase specific activity and total protein content were assayed.) by a HPLC protein pak 3005w gel filtration column. 102 o 3 -~s-w-—,-s-g~—o5vt - .—w ‘ .“IV_ V .v—‘v—g—fl . .v . . . ‘" ." l l l U Nd FIGURE 5.3 SDS-polyacrylamide gel electrophoresis of purified B—amylase. The B—amylase was electrOphoresed on 10% acrylamide gel and stained with Coomassie Brilliant blue R-ZSO. _B_- bio-gel filtration or _C_- combination of 3% ammonium sulfate and 20% ethanol; A— and D; standard marker proteins. The standards were: bovine serum albumin (66,000); egg albumin (45,000); glyceraldehyde 3-phosphate dehydrogenase (36,000); trypsinogen (24,000); carbonic anhydrous (20,000); and a-lactalbumin (14,000). 103 2000 “W1‘fi1111111111111[111111111111111111111111111111111111111[111111111 H AMMONIUM SULFATE .1 .4 O—e COMBINATlON /‘\ -‘ 'i :2 1500 ~ A c: j - V (I) ‘ .. ~ E — - 0") ~ Cl) — 1000 e _- 0 ’0 :5 ~ — L. O a Q — Q- ‘ .4 C d .1 O (D 500 —l .. E - . , . "l .1 .J 0 ‘ - -.. _ O 11111111111111111111111111111r111111111‘11111111111111111111111111111]r11T11111 0 20 40 60 80 100 120 140 l60 Time (Min) FIGURE 5.4 Change in mean particle size with time for B—amylase precipitated by a combination of 3% ammonium sulfate and 20% ethanol and by 50% ammonium sulfate alone. A Coulter Electronics Model N4 sub-micron particle analyzer with 200 seconds auto sampling time was used in all the experiments. 104 TABLE 5.2 A comparison of partial purification of B-amylase from C.thermosulfurogenes by various methods specific Protein Recovery Purification Step Activity Conc. in (%) Fold (U/mg) Supernatant (mg/m1) B—amylase 4.33 2.93 100 1 Cultured broth Concentration 28.6 3.25 92 6.60 by UF Precipitation 889 0.41 72 205 by Combination Precipitation 237 1.40 86 54.7 only by ammonium sulfate * Precipitation 125 0.49 21 28.9 only by ethanol * 20% ethanol and 3% ammonium sulfate precipitate 105 5.5 DISCUSSION The extracellular B-amylase produced by C thermosulfurogenes ‘was easily purified to near homogeneity with 72 percent activity recovery and with 205 purification fold by the combination of ethanol and ammonium sulfate precipitation. About 30 fold purification with 78 percent recovery was obtained in a simple batch precipitation step. The purity of the B-amylase is shown in figures 5.1 and 5.2, and ' compared with the highly pure B-amylase obtained by gel filtration chromatography (figure 5.2). These results suggest that only a small amount of impurity is present in the B-amylase after precipitation. The combination effect of salt and solvent is not well characterized but it was suggested that the opposite effects on the solubility of proteins makes it possible to adjust the solubility of a protein to a convenient value by balancing the solvent action of salt with the precipitating action of the organic solvent. Different amounts of salt and solvent have produced different results in precipitation (Table 5.1). Mean particles were stabilized by the presence of ammonium sulfate and increased in size for longer stirring (figure 5.4). This result is different from the ammonium sulfate precipitation. In summary, the combination of organic salt and solvent precipitation is a very efficient, simple, cheap, and short time purification process. Four times more purification fold can be obtained than precipitation by ammonium sulfate alone in the present case. In a different istudy, only 91 fold purification with 64 percent recovery was obtained for B-amylase partial purification by ethanol and followed by raw starch adsorption and soluble starch elution in a column (Saha and Zeikus, 1988). Comparing these results, the combination of ethanol and ammonium sulfate 106 precipitation is better for purification of B—amylase from low activity broth to higher activity. This is also a simple, fast, and low cost purification process. All these experiments were conducted at room temperature with different amounts of salts and solvents. The purification can be further optimized by conducting the precipitation at different temperature and pH. 107 5.7 LITERATURE CITED Bell, D. J., M. Hoare and P. Dunnill, "The formation of protein precipitates and their centrifugal recovery", Adv. Bichem., 1983, 26:1-72. Bonnerjea, J., Steven Oh, M. Hoare, and P. Dunnill, "Protein purification", Bio/Technology, 1986, 4954-58. Cohn, E. J., L. E. Strong, W. L. Hughes, Jr., D. J. Mulford, J. N. Ashworth, M. Melin, and H. L. Taylor, "Precipitation and properties of serum and plasma proteins", J. Amer. Chem. Soc., 1946, 68:459-75. Hoshino, M., Y. Hirose, K. Sano and K. Mitsugi, "Adsorption of microbial B-amylase on starch", Agr. Biol. Chem., 1975, 39, 12:2415-16. Hyun, H. H. and J. G. Zeikus, "General biochemical characterization of thermostable extracellular [3.amylase from Clostridium thermosulfurogenes", Appl. Environ. Microbiol., 1985, 49:1162-67. Hyun, H. H. and J. G. Zeikus, "Regulation and genetic enhancement of B-amylase production in Clostridium thermosufigrogenes", J. Bacteriol., 1985, 16421162—70. Laemmli, U. K., "Cleavage of structural protein during the assembly of the head of bacteriophage T4," Nature (London), 1970, 227:680-85. Lowry, P. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, "Protein measurement with the Folin phenol reagent", J. Biol. Chem., 1951, 193:265—75. Miller, G. L., "Use of dinitrosalicylic acid reagent for determination of reducing sugars", Anal, Chem. 1969, 31:426-428. Saha, B. C., G. J. Shen and J. G. Zeikus, "Behavior of a novel thermostable B-amylase on raw starch", Enzyme Microb. Technol., 1987, 9:598-601. Schubert, P. F., and R. K. Finn, "Alcohol precipitation of proteins: The relationship of denaturation and precipitation for catalase", Biotech. Bioeng., 1981, 23:2569-90. Ueda, S. and J. J. Marshall, "On the ability of pullulanase to stimulate the enzymatic digestion of raw starch", Carbohydrate Research, 1980, 84:196-8. Wilchek, M., T. Miran and J. Kohn, "Affinity chromatography", Methods. Enzymol, 1984, 1043-55. CHAPTER 6 SUMMARY AND CONCLUSIONS 6.1 Conclusions Free: From fermentation and concentration stgdies (Ch. 3): 1. A simple scale-up in B-amylase production has no influence in mass transfer rate or final B-amylase activity. 2. Production time of B-amylase in a batch fermenter is eight to twelve hours and can be reduced by inoculating with a seed culture at its exponential growth stage. 3. Concentration of B-amylase with 30,000 mwco UF membrane yields more than 85% activity recovery with ten fold purification for all four carbon sources used in the B-amylase production. 4. B—amylase produced by maltrin fermentation yields about 90% activity recovery with 100,000 MWCO UF membrane while B—amylase produced by maltose fermentation yields only about 50% activity recovery with the same membrane. Using a bigger MWCO membrane reduce the process time and cost a great deal. 108 1.92 . From precipitation gnd molecu_lgr weight estimation stgdies (Ch. 43 l. The mechanism of the precipitation of B-amylase with ammonium sulfate and ethanol depend on the fermentation carbon source. Fifty percent saturated ammonium sulfate or twenty volume percent ethanol is the optimum concentration for the recovery and purification of C.thennosuMgenes derived B~amylase for salt and organic solvent precipitation, respectively. Room temperature is the optimum temperature for 0amylase recovery and purification with both agents. Ammonium sulfate precipitation results in higher activity yield from fermentation broths with maltose as a carbon source. Starch dextrins present in the fermentation broth form a biospecific complex with B—amylase which can be easily precipitated with ethanol. Ethanol precipitation of B-amylase gives higher recoveries with starch dextrin containing broths and is a biospecific recovery process. Three purification steps are enough to purify the B-amylase to homogeneity with B—amylase specific activity of about 4,000 U/mg. Maluin fermented B-amylase has a higher effective molecular weight (228,000 daltons) clusters formed by B—amylase subunits. About 50,000 daltons molecular weight was obtained in denatured form with maltose and maltrin determined by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. 110 10. B-amylase purified by gel filtration shows higher specific activity compare to another highly purified B—amylases from Thermoactinomycel or from sweet potato. From combination of precipitation studies (Ch. 5): 1. B-amylase could be easily purified to near homogeneity by combination of 20% ethanol and 3% ammonium sulfate batch precipitation. 2. Combination of organic solvent and salt precipitation is a very efficient, simple and low cost purification process, and has great potential in protein purification. 6.2 Proposals for future research 1. More ultrafiltration studies are necessary with pure B-amylase to further understand the B-amylase-substrate complex fomration. This can be performed by adding known amount of dextrin into pure B—amylase produced with maltose and then concentrating with 100,000 MWCO UF membrane. The increase in recovery in the concentrate could be related to the complex formation. 2. More light scattering and turbidity studies are necessary to predict the agglomeration/break up mechanism in ethanol precipitated B-amylase (produced by maltrin fermentation) and in the combination of ethanol and ammonium sulfate precipitation. 111 3. Further studies are necessary to optimize temperature, pH, and initial B-amylase concentration for combination of salt and solvent precipitation. This will lead to a simple purification process of proteins. 4. The combination of precipitation techniques should be applied to other proteins. 112 TABLE A.1 Components used in fermentation to prodpce Baflylase Components NH4Cl KHZPO4 NazHPO,.7H20 MgC12.6H20 Trypticase Yeast Extract Vitamin Solution Trace Mineral FeSO, (2.5%) Resazurin Maltose/ maltrin Amount per Liter 1.0 gram 0.3 gram 2.1 gram 0.2 gram 10.0 gram 3.0 gram 10.0 ml 10.0 ml 0.25 III 1.0 ml 10.0 gram 113 TABLE A.2 RAW DATA ULTRAFILTRATION STUDIES #1 Concentration and purification of B-amylase with cells by ultrafiltration Volume Activity Protein Yield (ml) (U/ml) Conc. (%) (mg/ml) Original Broth 1520 13.0 4.15 100 Concentrate 220 80.0 4.07 90 Perrneate 1300 3.0 4.32 -- Fermentation Carbon Source : 1% maltose Membrane Size: 30,000 MWCO Cartridge Membrane Type: Diaflo Hollow Fiber (Amicon) Purification Fold: 7.0 114 TABLE A.3 RAW DATA ULTRAFILTRATION STUDIES #2 Concentration and purification of B-amylase with cells by ultrafiltration Volume Activity Protein Yield (ml) (U/ml) Conc. (%) (mg/ml) Original Broth 110,000 45.0 4.34 100 Concentrate 3,000 1396.0 8.18 85 Permeate 107,000 8. 1 ---- -- Fermentation Carbon Source : 1% maltose Membrane Size: 30,000 MWCO Cartridge Membrane Type: Diaflo Hollow Fiber (Amicon) Purification fold: 16.5 115 TABLE A.4 RAW DATA ULTRAFILTRATION STUDIES #3 Concentration and purification of B-amylase with cells by ultrafiltration Volume Activity Protein Yield (ml) (U/ml) Conc. (%) (mg/ml) Original Broth 11,000 26.7 4.46 100 Concentrate 1,200 216.0 3.45 '88 Permeate 9,800 3.0 4.12 -- Fermentation Carbon Source : 1% soluble starch Membrane Size: 30,000 MWCO Cartridge Membrane Type: Spiral (Amicon) Purification Fold: 10.5 116 TABLE A.5 RAW DATA ULTRAFILTRATION STUDIES #4 Concentration and purification of Bamylase with cells by ultrafiltration Volume Activity Protein Yield (ml) (U/ml) Conc. (%) (mg/ml) Original Broth 12,000 25.93 3.03 100 Concentrate 840 315.72 2.88 85 Permeate 11,160 --- ---- -- Fermentation Carbon Source : 1% Maltrin-40 Membrane Size: 30,000 MWCO Cartridge Membrane Type: Spiral (Amicon) Purification Fold: 12.7 117 TABLE A.6 RAW DATA ULTRAFILTRATION STUDIES #5 Concentration and purification of B—amylase by ultrafiltration Volume Activity Protein Yield (ml) (U/ml) Conc. (%) (mg/ml) Original Broth 8,500 27.8 4.19 100 Concentrate 740 271.4 3.59 85 Permeate 7,760 --- ---- -- Fermentation Carbon Source : 1% Maltrin-100 Membrane Size: 30,000 MWCO Cartridge Membrane Type: Spiral (Amicon) Purification Fold: 11.4 118 TABLE A.7 RAHATA ULTRAFILTRATION smpms #6 Concentration and purification of B-amylase with cells by ultrafiltration Volume Activity Protein Yield (ml) (U/ml) Conc. (%) . (mg/ml) Original Broth 2,000 213.3 3.81 100 Concentrate 200 2080.0 8.39 90 Permeate 1,800 19.0 2.70 Fermentation Carbon Source : 1% Maltrin-lOO Membrane Size: 100,000 MWCO Cartridge Membrane Type: Diaflo Hollow Fiber (Amicon) Purification Fold: 4.5 119 TABLE A.8 RAW QATA ULTRAFILTRATION STUDIES #7 Concentration and purification of B-amylase with cells by ultrafiltration Volume Activity Protein Yield (ml) (U/ml) Conc. (%) (mg/ml) Original Broth 21,000 216.38 3.79 100 Concentrate 1,600 2496.0 16.70 86 Permeate 20,000 ---- ---- ~- Fermentation Carbon Source : 1% Maltrin-100 Membrane Size: 30,000 MWCO Cartridge Membrane Type: Spiral (Amicon) Purification Fold: 2.6 120 TABLE A.9 RAW DATA ULTRAFILTRATION STUDIES #8 Concentration and purification of B—amylase with cells by ultrafiltration Volume Activity Protein Yield (ml) (U/ml) Conc. (%) (mg/ml) Original Broth 1027 6.79 3.26 100 Concentrate 127 19.22 3.60 35 Permeate 900 3.97 2.70 Fermentation Carbon Source : 1% maltose Membrane Size: 100,000 MWCO Cartridge Membrane Type: Diaflo Hollow Fiber (Amicon) Purification Fold: 2.7 121 TABLE A.10 RAW DATA ULTRAFILTRATION STUDIES #9 Concentration and purification of B—amylase with cells by ultrafiltration Volume Activity Protein Yield (ml) (U/ml) Conc. (%) (mg/ml) Original Broth 2075 34.0 3.23 100 Concentrate 225 150.0 3.18 50 Permeate 1850 10.0 4.03 Fermentation Carbon Source : 1% maltose Membrane Size: 100,000 MWCO Cartridge Membrane Type: Diaflo Hollow Fiber (Amicon) Purification Fold: 4.7 122 TABLE A.11 RAW DATA ULTRAFILTRATION STUDIES #10 Concentration and purification of B-amylase with cells by ultrafiltration Volume Activity Protein Yield (ml) (U/ml) Conc. (%) (mg/ml) Original Broth 94,000 34.0 3.21 100 Concentrate 6,000 471.0 3.34 88 Permeate 88,000 3.0 ---- -- Fermentation Carbon Source : 1% maltose Membrane Size: 30,000 MWCO Cartridge Membrane Type: Spiral (Amicon) Purification Fold: 14.0 123 TABLE A.12 RAW DATA ULTRAFILTRATION STUDIES #10 Concentration and purification of B-amylase with cells by ultrafiltration Volume Activity Protein Yield (ml) (U/ml conc. (%) supernatant) (mg/ml) Original Broth 2042 34.0 --- 100 Concentrate 192 288.0 --- 80 Permeate 1850 10.0 --- -- Fermentation Carbon Source : 1% maltose Membrane Size: 30,000 MWCO Cartridge Membrane Type: Diaflo Hollow Fiber (Amicon) Purification Fold: 7.6 124 TABLE A.13 RAW DATA PRECIPITATION STUDIES #1 Precipitation of B-amylase by 50% saturated ammonium sulfate or 20 % (v/v) ethanol Activity Protein Purific- Yield (U/ml conc. cation (%) supernatant) (mg/ml) Ratio Concentrated 135.2 3.70 1.0 100 B—amylase Concentrate 254.8 1.07 6.47 94 (ammonium sulfate) Concentrate 61.5 0.49 3.43 23 (ethanol) Fermentation Carbon Source : 1% maltose Original volume: 50.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm PRECIPITATION STUDIES #2 125 TABLE A.14 RAW DATA Precipitation of B-amylase by 50% saturated ammonium sulfate or 20 % (v/v) ethanol Activity Protein Purific- Yield (U/ml conc. cation (%) supernatant) (mg/ml) Ratio Concentrated 332. 1 3.60 1.0 100 B—amylase Concentrate 149.8 0.72 2.25 15 (ammonium sulfate) Concentrate 659.4 1 .30 5.50 80 (ethanol) Fermentation Carbon Source : 1% maltrin Original volume: 75.0 ml Sttirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm Purification ratio = (final enzyme activity (U/mg)/ initial enzyme activity (U/mg) 126 TABLE A.15 RAW DATA PRECIPITATION STUDIES #3 Precipitation of B-amylase by 50% saturated ammonium sulfate or 20 % (v/v) ethanol Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 871.2 22275.0 1.0 100 B—amylase Concentrate 125.7 21875.0 7.0 98 (ammonium sulfate) Concentrated 594.0 15186.0 1.0 100 B-amylase Concentrate 35.1 6636.0 7.2 ‘ 45 (ethanol) Fermentation Carbon Source : 1% maltrin Original volume: 50.0 ml for ammonium sulfate ppt. 75.0 ml for ethanol ppt. Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm Purification ratio = (final enzyme activity (U/mg)/ initial enzyme activity (U/mg) 127 TABLE A.16 RAW DATA PRECIPITATION STUDIES #4 Precipitation of B-amylase by 20 % (v/v) ethanol at different temperatures. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 141.0 18802.0 1.0 100 B—amylase Precipitate 18.7 6908.0 2.75 37 at 4°C Precipitate 13.7 4021.0 2.25 21 at 25°C Precipitate 15.7 2626.0 1.24 14 at 35°C Precipitate 53.9 6578.0 0.90 34 at 5°C Supernatant 1 16.5 10149.0 ---- -- at 4°C Supernatant 107.5 12628.0 ---- -- at 25°C I Supernatant 124.9 11162.0 ---- -- at 35°C Supernatant 58.2 13260.0 ---- -- at 5°°C Fermentation Carbon Source : 1% maltose Original volume: 75.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm 128 TABLE A.17 RAW DATA PRECIPITATION STUPIES #5 Precipitation of B-amylase by 20 % (v/v) ethanol at different temperatures. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 205.0 29823.0 1.0 100 B-amylase Precipitate 21.5 9345.0 2.90 31 at 4°C Precipitate 28.4 5620.0 1.40 19 at 25°C Precipitate 24.8 7850.0 2.20 23 at 35°C Precipitate 49.8 6986.0 0.54 34 at 50°C Supernatant 165.9 21819.0 ---- -- at 4°C Supernatant 167.3 22584.0 ---- -- at 25°C Supernatant 158.9 23599.0 ---- ~- at 35°C Supernatant 99.4 19782.0 ---- -- at 50°C Fermentation Carbon Source : 1% maltose Original volume: 75.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm PRECIPITATION STUDIES #6 129 TABLE A.18 RAW DATA Precipitation of B—amylase by 20 % (v/v) ethanol at different temperatures. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 253.9 25387.0 1.0 100 B-amylase Precipitate 53.2 1 1948.0 2.2 47 at 4°C Precipitate 57.0 22762.0 4.0 90 at 25°C Precipitate 76.1 18180.0 2.4 72 at 35°C Precipitate 46.7 12850.0 2.7 57 at 50°C Supernatant 206.8 7665.0 -- at 4°C Supernatant 195.4 3523.0 ---- -- at 25°C Supernatant 187.0 6390.0 ~--- - at 35°C Supernatant 193.4 7247.0 ---- -- at 50°C Fermentation Carbon Source : 1% maltrin Original volume: 75.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm 130 TABLE A.19 RAW QATA PRECIPITATION STUDIES #7 Precipitation with 50 % sat. ammonium sulfate at different temperatures. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 187.6 15204.0 1.0 100 B-amylase Precipitate 89.4 9395.0 1.3 62 at 4°C Precipitate 80.2 8147.0 1.3 54 at 25°C Precipitate 91.6 9883.0 1.3 65 at 35°C Precipitate 100.6 14040.0 1.7 92 at 50°C Supernatant 82.7 8997.0 ---- -- at 4°C Supernatant 82.1 8323.0 ~--- -- at 25°C Supernatant 83.6 8273.0 ---- -- at 35°C Supernatant 81.6 7140.0 ---- -- at 50°C Fermentation Carbon Source : 1% maltrin . Original volume: 50.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm 131 TABLE A.20 RAW DATA PRECIPITATION STUDIES #8 Precipitation of B~amylase by 50 % sat. ammonium sulfate at different temperatures. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 187.6 15204.0 1.0 100 B-amylase Precipitate 74.1 10009.0 1.7 66 at 4°C Precipitate 75.3 9027.0 1.5 59 at 25°C Precipitate 80.1 10170.0 1.6 67 at 35°C Precipitate 78.2 1 1237.0 1.8 78 at 50°C * Precipitate 83.6 . 13068.0 1.9 86 at 4°C * Precipitate 86.6 10562.0 1.5 70 at 25°C Fermentation Carbon Source : 1% maltrin Original volume: 50.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm ' Precipitation was conducted in a baffled reactor 132 TABLE A.21 RAw DATA PRECIPITATION STUpIES #9 Precipitation of B—amylase by 50 % sat. ammonium sulfate at different temperatures. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 155.6 7367.5 1.0 100 B-amylase Precipitate 56.1 5072.5 2.0 69 at 25°C Precipitate 43.3 7043.0 3.5 96 at 35°C Precipitate 42.0 7246.0 3.7 98 at 50°C Fermentation Carbon Source : 1% maltrin Original volume: 50.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm 133 TABLE A.22 RAW DATA PRECIPITATION STUDIES #10 Precipitation of [ii-amylase by 20 % (vN) ethanol at different temperatures. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 259.5 14897.0 1.0 100 B-amylase Precipitate 50.9 8647.3 3.0 58 at 4°C Precipitate 29.8 10617.9 6.2 72 at 25°C Precipitate 36.4 10080.5 4.8 68 at 40°C Precipitate 65.4 9788.7 2.6 66 at 50°C Fermentation Carbon Source : 1% maltrin Original volume: 75.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm PRECIPITATION STUDIES #11 TABLE A.23 RAW DATA 134 Precipitation of B-amylase by 50 % saturated ammonium sulfate at different temperatures. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 340.0 12721.0 1.0 100 B—amylase Precipitate 241.0 12561.0 1.4 98.7 at 4°C Precipitate 265.6 1 1465.9 1.3 99.0 at 20°C Precipitate 268.4 1 1465.5 1.2 90.0 at 40°C Precipitate 274.2 1 1232.7 1.1 88.0 at 50°C Fermentation Carbon Source : 1% maltose Original volume: 50.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 rrrin at 12,000 rpm PRECIPITATION STUDIES #1; 135 TABLE A.24 RAW DATA Precipitation of B—amylase by 50 % saturated ammonium sulfate at different temeratures. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 201.8 7345.0 1.0 100 B-amylase Precipitate 170.4 6878.0 1.1 93 at 4°C Precipitate 174.4 7291.0 1.2 99 at 25°C Precipitate 185.5 7156.0 1.1 97 at 35°C Precipitate 180.0 5856.0 1.0 79 at 50°C Fermentation Carbon Source : 1% maltose Original volume: 50.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm 136 TABLE A.25 RAW DATA PRECIPITATION STUDIES #13 Precipitation of B—amylase by different concentration of ethanol at room temperature. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 331.3 31275.0 1.0 100 B-amylase Precipitate 54.0 7884.0 1.8 25 with 10% ethanol Precipitate 57. 1 8394.0 1.8 27 with 20% ethanol Precipitate 68.0 8798.0 1.6 28 with 30% ethanol Precipitate 105.0 8629.0 1.0 28 with 40% ethanol Precipitate 150.4 7069.0 0.6 23 with 50% ethanol Supernatant, 10% 285.5 20767.0 ---- -- Supernatant, 20% 275.3 20232.0 ---- - Supernatant. 30% 267.1 23304.0 ---- - Supernatant, 40% 204.1 22464.0 --- - Supernatant, 50% 106.1 25082.0 --- - Fermentation Carbon Source : 1% maltose Original volume: 75.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm 137 TABLE A.26 RAW DATA PRECIPITATION STUDIES #14 Precipitation of B—amyalse by different concentration of ethanol at room temperature. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 253.9 25387.0 1.0 100 B-amylase Precipitate 29.4 7621.0 2.6 30 with 10% ethanol Precipitate 56.9 22762.0 4.0 90 with 20% ethanol Precipitate 98.3 1 1745.0 1.8 70 with 30% ethanol Precipitate 75.0 18245.0 2.5 72 with 40% ethanol Precipitate 70.1 12745.0 1.8 50 with 50% ethanol Supernatant. 10% 223.1 15292.0 ---- -- Supernatant, 20% 195.4 3523.0 --- -- Supernatant, 30% 204.0 6576.0 --- -- Supernatant, 40% 207.0 9477.0 --- -- Supernatant. 50% 213.1 9040.0 --- -- Fermentation Carbon Source : 1% maltrin Original volume: 75.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm 138 TABLE A.27 RAW DATA PRECIPITATION STUDIES #15 Precipitation of B-amylase by different concentration of ethanol at room temperature. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 259.5 14897.0 1.0 100 B-amylase Precipitate 29.8 10618.0 6.2 72 with 20% ethanol Precipitate 53.8 13019.0 4.2 87 with 30% ethanol Precipitate 90.5 10952.0 4.2 74 with 40% ethanol Precipitate 34.5 13567.0 7.0 91 with 20% ethanol 4 hrs. stirring Precipitate 61.8 13548.0 3.6 50 with 30% ethanol 4 hrs. stirring Fermentation Carbon Source : 1% maltrin Original volume: 75.0 ml Stirring time during precipitation: 120 min Centrifucation time: 30.0 min at 12,000 rpm PRECIPITATION STUDIES #16 TABLE A.28 RAW DATA 139 Precipitation with different concentration of ethanol at room temperature. Total Total Purific- Yield Protein Activity cation (%) (mg) (Units) Ratio Concentrated 220.9 13919.0 1.0 100 B-amylase Precipitate 17.8 2562.0 1.5 19 with 10% ethanol ' Precipitate 26.4 1 1794.0 7.1 85 with 20% ethanol Precipitate 32.2 12410.0 5.4 89 with 30% ethanol Precipitate 36.5 1 1482.0 5.0 82 with 40% ethanol Precipitate 37.9 1 1219.0 4.7 80 with 50% ethanol Fermentation Carbon Source : 1% soluble starch Original volume: 75.0 ml Stirring time during precipitation: 120 min Centriufcation time: 30.0 min at 12,000 rpm "illilllilllililllllllllil“