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Studies of [alpha]-galactosidases isolated from Clostridium perfringens and waste lager yeast (Saccharomyces… Durance, Timothy Douglas 1984

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C c STUDIES OF a-GALACTOSIDASES ISOLATED FROM CLOSTRIDIUM PERFRINGENS AND WASTE LAGER YEAST (SACCHAROMYCES CARLSBERGENSIS) A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF FOOD SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1984 ® Timothy Douglas Durance, 1984 by TIMOTHY DOUGLAS DURANCE MASTER OF SCIENCE i n In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. DEPARTMENT OF FOOD SCIENCE The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: September. 1984 Abstract This study is divided into two parts. Part 1 describes the partial purification and characterization of a Clostridium perfringens o-galactosidase while part 2 describes the isolation and characterization of a-galactosidase from waste lager yeast (Saccharomyces carlsbergensis, also known as S. uvarum). Of 21 strains of C. perfringens tested, 10 utilized either raffinose or melibiose while 2 utilized both sugars. Spore production in Duncan Strong medium was superior in the presence of raffinose as opposed to starch in 12 of 21 strains. One strain of C. perfringens (M34) yielded 1.2 units of o-galactosidase/g washed cells. This enzyme had an isoelectric point of 5.6 and a pH optimum for hydrolysis of p-nitrophenyl a-D-galactopyranoside (PNPG) of 6.3. Molecular weight of the native protein was 96,000. Monomer molecular weight appeared to be 46,000. Km was 0.20 ±0.02mM PNPG. Heat stability of the enzyme at 45°C decreased as purity increased. This trend was only partially reversed by the addition of 2-mercaptoethanol, NADH, cysteine, and/or bovine serum albumin to the reaction mixture. Waste lager yeast was found to contain 27 Units of a-galactosidase /g yeast cells (dry weight). This activity was resolved into 3 active peaks by DEAE cellulose chromatography. Subsequent gel filtration indicated molecular weights of the a-galactosidase in peaks A, B and C to be 118,000, 95,000, and 65,000 respectively. The isoelectric point of all three forms of the enzyme was 4.4 ±0.1. The enzyme of peak C was shown to have a pH optimum of 4.5. Km values (± standard errors) were 2.54 ±0.32 mM p-nitrophenyl PNPG and 21.1 ±1.8 mM raffinose. ii Table of Contents Abstract i i List of Tables v i List of Figures .V i i i Acknowledgements x I. Literature Review 1 A. Microbial o-Galactosidases 1 1. Distribution 1 2. Localization of c-GAL in Prokaryotic and Eukaryotic Cells 1 3. Isolation and Purification 4 4. Physical Properties 5 5. Kinetic Properties of o-GALs '. 8 B. o-GAL and Invertase systems of Saccharomyces carlsbergensis 15 C. Flatulence and Legume Consumption 17 D. The Application of Microbial o-GAL to Food Processing .21 1. o-GAL Treatment of Soy Milk 21 2. c-GAL Treatment of Sugar Beet Molasses 22 E. DETERMINATION OF INITIAL VELOCITIES. Km AND Vmax OF ENZYME CATALYZED REACTIONS 23 1. Initial Velocity 23 2. Km and Vmax 24 F. Sporulation of Clostridium perfringens 27 II. Purification and Characterization of the c-Galactosidase of Clostridium perfringens 29 A. Introduction 29 B. Materials and Methods 29 1. Microorganisms .^ 30 2. Purification of o-GALCp 31 i i i 3. E n z y m e A c t i v i t y 32 4. E lec t rophores i s 32 5. Isoelectr ic Focus ing 33 6. Thermal Stabi l i ty 33 7. Substrate A f f i n i t y 34 8. Protein Determinat ions 34 C. Results and D i s c u s s i o n 34 1. Strain Se lec t i on 34 2. Sporu la t ion 34 3. Y ie lds of a - G a l a c t o s i d a s e 36 4. Pur i f i cat ion of o - G A L C p 38 5. pH O p t i m u m 38 6. Mo lecu lar Weights 43 7. Isoelectr ic Point 43 8. A f f i n i t y for P N P G 43 9. A c t i v a t i o n of o - G A L C p by Var ious C o m p o u n d s .43 10. Heat Stabi l i ty 49 D. C o n c l u s i o n s 52 III. Isolation and Character izat ion of the a - G a l a c t o s i d a s e of W a s t e Lager Yeast (Saccharomyces carlsbergensis) 53 A . Introduction 53 B. Mater ia ls and Methods .'. 54 1. Brewers Yeast 54 2. Crude Extract 54 3. Isopropanol Prec ip i tat ion 54 4. Sephadex G100 Gel F i l trat ion Chromatography 54 5. A n i o n Exchange Chromatography 55 i v 6. Sephacry l S400 Gel Fi l trat ion Chromatography 55 7. Protein Measurements 55 8. o - G a l a c t o s i d a s e A c t i v i t y [ 55 9. Molecular Weight Es t imat ion 56 10. Isoelectric Focus ing 57 11. Substrate A f f i n i t y 57 12. Determinat ion of pH Opt imum 58 C. Results and D i s c u s s i o n 58 1. Y ie lds 58 2. Pur i f icat ion 58 3. Sod ium D o d e c y l Sul fate Po lyac ry lamide Gel E lect rophores is 60 4. Isoelectric Focus ing 64 5. Mult iple Molecu lar Fo rms of c - G A L S c 64 6. pH Opt imum „ 66 7. Substrate A f f i n i t y 66 D. Conc lus ions ~ ; 72 IV. References ~ 73 v List of Tables Table Page 1. Microbial sources of a-GAL (Ulezlo and Zaprometova, 1982) 2 2. Microbial o-GAL's which have been studied in highly purified preparations 6 3. Physical properties of some microbial o-GAL's; molecular weights (MW) and isoelectric points (pi) 7 4. Substrate specificity of o-GAL from Vic/a faba: aglycon specificity (Dey and Pridham, 1972) 9 5. Kinetic properties of some microbial o-GAL's 11 6. Kinetic properties of purified o-GAL of Saccharomyces carlsbergensis based on data from Lazo et al., (1978) 18 7. Production of acid and gas from carbohydrates by strains of C. perfringens 35 8. Spore counts of C. perfringens strains grown in Duncan Strong sporulation medium with starch or raffinose as the carbon source (thousands of spores/mL) 37 9. Effects of purification steps on specific activity and yield of o-GALCp 39 10. Effects of various compounds on o-GALCp activity at 30°C and 45°C 48 11. Purification of o-GALSc by 2-propanol precipitation, G100 Sephadex gel filtration, DEAE cellulose and Sephacryl S400 gel filtration chromatography 59 12. Kinetic parameters of c-GALSc (± standard errors) and residual standard errors (RSE), computed with the program of Oestreicher and V i Pinto (1983) using initial velocities estimated by fixed time assays or derived from experimentally determined curves 68 13. Results of curve fitting to a-GALSc kinetic data at various initial substrate concentrations 71 v i i List of Figures Figure p a g e 1. Chromatography of o-GALCp on DEAE cellulose: absorbance. (•); a-galactosidase activity, ( A ) ; sodium chloride concentration. (. .) 40 2. SDS-polyacrylamide gel electrophoresis of a-GALCp and' protein standards; A, a-GALCp peak from Sephacryl S400 chromatography; B, catalase; C, bovine serum albumin (polymerized) 41 3. pH optimum of a-GALCp at 30°C. Each point presents the average of duplicate determinations 42 4. Gel filtration chromatography of a-GALCp and molecular weight standards on Sephacryl S400 44 5. Electrophoretic mobility of a-GALCp and molecular weight standards on SDS- polyacrylamide gel 45 6. Lineweaver Burk plot of a-GALCp kinetic data of PNPG hydrolysis at pH 6.5 and 40°C: V0=velocity (uM/min); S = substrate concentration (mM) 46 7. Temperature optimums of a-GALCp in crude extract (•) and in active fractions of Sephacryl S400 chromatography (•). Each point represents the average of duplicate determinations 50 8. Thermal stability of a-GALCp. incubated at various temperatures for 15 min, then assayed at 35°C 51 9. DEAE cellulose chromatography of a-GALSc: a-galactosidase activity, (•); absorbance. (•); sodium chloride gradient, (••-). A, B, and C indicate areas of a-galactosidase activity. 61 10. Gel filtration of o-GALSc-A, a-GALSc-B, a-GALSc-C and protein v i i i standards on Sephacryl S400 62 SDS-polyacrylamide gel electrophoresis of o-GALSc fractions and bovine serum albumin: A, crude extract; B, o-GALSc peak from Sephadex G100 column; C, o-GALSc-A from DEAE cellulose column; D, o-GALSc-B from DEAE cellulose column; E, a-GALSc-C from DEAE cellulose column; F, bovine serum albumin (polymerized) 63 The effect of pH on activity of o-GALSc-C with PNPG (•) and raffinose (o) as substrates. Each point represents the averge of duplicate determinations 67 Hydrolysis of PNPG by o-GALSc-C at 40°C, pH 4.5, and 5 different initial PNPG concentrations 70 Acknowledgements I wish to express my gratitude to my supervisor, Dr. B.J. Skura, for his encouragement, guidance, and assistance throughout the course of this study. I would also like to thank my graduate committee, Dr. S. Nakai, Dr. W.D. Powrie, and Dr. P.M. Townsley for their valuable suggestions and assistance. x I. LITERATURE REVIEW MICROBIAL g-GALACTOSlPASES 1. DISTRIBUTION, The earliest references in the scientific literature to o-galactosidases (o-D-galactoside galactohydrolases (E.C.3.2.1.22)) date back to 1895 when Bau and Fisher and Lindner isolated an enzyme from a bottom fermenting brewers yeast which hydrolyzed melibiose. Weidenhagen" (1928) examined the activity of this enzyme against various sugars and suggested that the term "a-galactosidase" (a-GAL) was more appropriate than the previous term "melibiase". Since that time numerous bacteria, actinomycetes, yeasts, and filamentous fungi have been shown to produce a-galactosidases. Many of these enzymes are listed in Table 1. 2. LOCALIZATION OF o-GAL IN PROKARYOTIC AND EUKARYOTIC  CELLS The question of the cellular location of a-GAL in both bacteria and fungi has received some attention because of the bearing location has on purification techniques and on the mechanism of a-galactoside metabolism. In general, the bacterial a-GALs appear to be intracellular. In the case of the Escherichia coli D1021 plasmid encoded a-GAL, Schmid and Schmitt (1976) determined that the enzyme is active within the cytoplasmic membrane. a-Galactoside sugars are transported into the cell by specific permeases prior to hydrolysis. Akiba and Horikoshi (I978) examined the possibility that 1 Table 1. Microbial sources of a-GAL (Ulezlo and Zaprometova, 1982). Bacteria and Actinomycetes Filamentous Fungi Yeasts A erabacter aenogenes Bacillus ceneus B. deltrukii B. steanotherm ophilus Bacteriodes fragilis C lostridium perfringens C. m aebashi 0 iplococcus pneum onia Escherichia coli L actobaa'llus spp. M icrococcus spp. Pseudom onas atlantica Salmonella typhimurium Streptococcus bovis Streptom yces fnadiae A ^xrgillus niger A. fum igatus A. jeanseim ei A. niger A. orysae A. rubenscens A. saiatoi A. terreus A. terricda 4. wentu Cephalosporium acremonlum Circinella muscae W ortierella vinacea Penidllium canescens P. davforme C andida guillerm ondii Pichia gjillierm ondii Saccharom ycetes carlsbergensis S. cereviaae S. hiepenas S. italicus var. melibiosi Schisosaccharom yces japonicus S. malidevoraus S. octoaiorus Schwanniomyces alluvius oiivaoeus rces'gpinus P. cydcpium P. dupontii P. frequentans P. janth P. paxSUus P. piacarum P. thorn ii 4 the a-GAL of a Micrococcus strain might be located in the periplasmic space by attempting to release the enzyme with freeze-thaw treatments and with lysozyme treatment, both with negative results. They also failed to inactivate the enzyme of whole cells with diazo-7-amino-1,3-naphthalene disulfonic acid, again indicating that the enzyme is intracellular. A study of the a-GAL of Pseudomonas atlantica indicated that the active enzyme was intracellular, although an inactve form of the enzyme was detected in the periplasmic space (Day et al., I975). In Bacteroides fragi/is, most of the a-GAL activity was associated with the cells but some activity could be recovered from the cell free supernatant (Berg et al., 1980). The possibility of periplasmic location was not examined. In the limited number of cases which have been studied the a-GAL of fungi appear to be at least partially extracellular. The enzyme can be recovered from the mycelium free filtrates of Mortierella vinacea (Suzuki et al., 1969). The a-GAL of Aspergillus niger is secreted from the mycelia in a similiar fashion (Adya and Elbein, 1977). At least one form of a-GAL has been shown to be extracellular in the yeasts Saccharomyces cerevisiae and S. carlsbergensis (Martinez et al., 1982 ; Lazo et al., 1977). Active enzyme, however, can also be found within the cytoplasmic membrane. 3. ISOLATION AND PURIFICATION Considering the number of a-GALs which have been identified, relatively few have been extensively purified. Those which have been 5 purified are listed in Table 2. These studies have shown that standard enzyme purification methods, including solvent precipitation, ammonium sulfate precipitation, DEAE cellulose ion exchange chromatography, and gel filtration can be successfully employed with a-GAL. Recently, affinity chromatography has been successfully used for rapid purification of o-GAL (Harpaz et al., 1974 ; Mapes and Sweeley, 1973). The adsorbents used were Sepharose or succinoylaminoalkyi agarose in conjunction with the ligands o-D-galactopyranosyl amine and p-aminophenyl-melibioside respectively. 4. PHYSICAL PROPERTIES o-GALs from different microbial sources vary greatly in their physical properties (Table 3). Molecular weights have been recorded from 45,000 for the a-GAL of Aspergillus niger to 500,000 for the a-GAL of Pennici 11 ium duponti. The presence of polymeric structure has been demonstrated, both in bacteria and fungi, in some cases. Although isoelectric points (pi) have been determined in only a few cases, pi values have always been between 3.5 and 6.5 (Table 3). All fungal a-GALs which have been examined for carbohydrate content have been confirmed to be glycoproteins; these include the a-GALs of Aspergillus niger, Mortierel la vinacea, and Saccharomyces carlsbergensis (Adya and Elbein, 1977 ; Suzuki et al., 1970 ; Lazo et al., 1977). No bacterial a-GALs have been reported to be glycoproteins. Table 2. Microbial a-GAL's which have been studied in highly purified preparations. Organism Reference E scherichia coli Bacillus stearotherm ophitlus A spergillus niger C crticum rolfai Saccharom yces caridtergens's Schmid and Schmitt, (1976). Pederson and Goodman, (1980). Bahl and Agrawal, (1969). Adya and Elbein, (1977). Kaji and Yoshihara, (1972). Lazo et al., (1977). Table 3. Physical properties of some microbial a-GALs; molecular weights (MW) and isoelectric points (pi). Organism Monomer MW Native MW pi Reference Bacillus sp 312,000 S . stearotherw ophillus 81,000 280,000 84,000 325,000 Bacteroides fnagilis 125,000 £. coli D1021 plasmid 82,000 329,000 £. coli K12 200,000 M/cnococoys sp 367,000 Aspergillus niger 45,000 A . saitoi 290,000 A . auamori 130,000 Penicillium duponti 500,000 Saccharomyces caiisbergenss 100,000 300,000 Akiba and Horikoshi, (1976) Pederson and Goodman, (1980) 6.2 Berg et al., (1980) 5.1 Schmid and Schmitt, (1976) Burstein and Kepes, (1971) Akiba and Horikoshi, (1976) Adya and Elbem, (1977) Sugimoto and van Buren, (1970) McGhee et al., (1978) Arnaud et al., (1976) 3.6 Lazo et al., (1977) 90,000 8 5. KINETIC PROPERTIES OF o-GALS A number of kinetic properties of o-GAL are important for identification of the enzymes, for understanding the physiological functions of the enzymes, and for assessing the suitability of o-GALs for practical applications. These include substrate specificity, pH optimum, temperature optimum, and the reaction parameters Km and Vmax. a. Substrate specificity a-GALs catalyze the hydrolysis of o-galactosyl bonds following the general formula : ,— »v^\ , (1) c - R 4- R'OH ^ Y_/-0-R -f ROH O H O H R'OH is commonly water but both R and R' can be aliphatic or aromatic groups. As a rule the glycosidases are quite specific. A change in configuration of the hydrogen or hydroxyl group's at any single carbon of the glycoside may be sufficient to severely retard or entirely eliminate activity. Two factors are particularily important in a-GAL substrates; first the pyranose ring must be present; second, the configurations of the H and OH groups at carbons 1, 2, 3, and 4 must be similiar to D-galactose (Ulezlo and Zaprometova, 1982). The C6 carbon is less significant (Dey and Pridham, 1972). Thus a-D-fucosides and 0-L-arabinosides are often hydrolyzed by a-GALs. The nature of the aglycon group (corresponding to R in equation 1) also effects the affinity of the enzyme for the substrate. This trait has been extensively studied in some plant a-GALs. The specificity of a-GAL of V/c/a faba is outlined in Table 4. It is Table 4. Substrate specificity of o-GAL from Vida faba ; aglycon specificity. (Dey and Pridham, 1972). Substrate Vmax (umoles/min/mg protein) Km (mM) Methyl a-D-galactoside 1.66 7.13 Ethyl a-D-galactoside 1.66 8.93 n-Propyl a-D-galactoside 2.20 6.13 Phenyl a-O-galactoside 20.30 1.11 o-Cresyl a-D-galactoside 26.00 1.33 m-Cresyl a-D-galactoside 24.30 1.38 p-Cresyl a-O-galactoside 23.00 1.54 o-Nitrophenyl a-D-galactoside 42.10 1.14 m-Nitrophenyl a-D-galactoside 5.86 10.0 p-Nitrophenyl a-D-galactoside 25.53 0.38 Melibiose 2.54 0.96 Raffinose 28.40 4.00 Stachyose 9.00 7.50 10 noteworthy that the Km value corresponding to PNPG is lowest, suggesting a high affinity for this substrate. High affinity for PNPG has been noted for many a-GALs (Dey and Pridham, 1972). PNPG is the most widely used substrate for routine a-GAL assays because of this high affinity combined with the fact that the hydrolysis product, p-nitrophenol, can be easily quantitated spectrophotometrically. The Km values of most a-GALs for melibiose are similiar to or slightly higher than their Km for PNPG. The Michaelis-Menton constant for raffinose is often considerably higher again than that of melibiose. b. pH Optimum The pH optimum of some microbial a-GALs are listed in Table 5. The bacterial enzymes tend to be most active close to neutrality while fungal a-GALs have optimums in the range of pH 4 to 5. In some instances however, the enzymes may be active over a wide range of H ion concentration. For example the a-GAL of Cladiosporium cladiosporoides retains >80% of maximum activity from pH 3.8 to pH 6.3 (Cruz et a I., 1981). The a-GAL of Saccharomyces carlsbergensis exhibits >80% relative activity from pH 3.8 to 5.2 (Lazo et al., 1978). c. Thermal Stability The temperature optimum for most microbial o-GALs are in the 37-40°C range. (Ulezlo and Zaprometova, 1982). There are, however, some noteworthy exceptions, several of which have been studied in detail because of the possibility of enhanced practical value of thermophillic enzymes. Bacillus stearothermophiIlus produces Table 5. Kinetic properties of some microbial o-GAL. Organism pH Optimum Temperature Km Substrate Reference Optimum (' C) (mM) E. coli K12. 7.5 3 PNPG Burstein & Kepes,197 1 10 Mel E. coli D1021 plasmid 7.2 0.14 PNPG Schmid & Schmitt, 1976 3.2 Mel E. coll com m unior Bacillus stearotherm cphilus 6.8 37 isozyme 1. isozyme 2. 73 69 0.1 1 PNPG 2.32 Mel Kawamura et al. 1976 0.47 19 0.53 PNPG Mel PNPG Pederson & Goodman. 1980 4 5 Mel Micrococcus X>. 7.5 35 Bacillus 32 6.5 40 A •pergfllus niger 4-4.5 A. awamcri 5.0 50 M ortlenella vtnaoea 4.0 50 Cladla&crtum dadla&oroiOes 5.0 65 0.47 ONPG 1.5 Mel 12.6 Raf 1.0 ONPG 7.9 Mel 24.1 Raf 0.18 PNPG 0.50 Raf 6.0 PNPG 30 Mel 36 Raf 0.4 PNPG 0.39 Mel 1.83 Raf Akiba & Horikoshi, 1976. Akiba & Horikoshi, 1976 Adya & Elbein. 1977 McGhee et al 1978 Suzuki et al, 1969. 1970 Cruz el al, 1981. Sacchanomyces carlstxrgenss 4.5 6.0 PNPG Lazo et al., 1977 6.0 Mel 135 Raf 1. PNPG = p-nitrophenyl a-D-galactosidase; Mel = melibiose; Raf = raffinose. 14 a -GALs which are stable for more than 30 min at 65°C (Pederson and Goodman, 1980). PeniciIlium dupontii and Cladiosporium cladiosporoides a-GAL 's have simil iar stabil ity (Arnaud et al., 1976; Cruz et al., 1981). Some other a -GALs have been found to be remarkably unstable. The a -GAL of Escherichia coli subsp communior as described by Kawamura et al., (1976) was signif icantly deactivated by 15 min incubation at temperatures of 25°C and above. This effect was particularily apparent at low protein concentrations. Burstein and Kepes (1971) made similiar observations about an E. coli K12 a -GAL , an enzyme which on the basis of kinetic data, appears to be different than the enzyme described by Kawamura et al., (1976). Several compounds have been used to improve the apparent activity of various a -GALs . Pederson and Goodman (1980) found that 1.5 mg/mL bovine serum albumin or gelatin improved the heat stabil ity of the B. stearothermophi11 us a -GAL . Burstein and Kepes (1971) found that NAD, NADH, 2-mercaptoethanol, and crude cell free extracts (without a -GAL act ivity) all improved the apparent activity of the E. coli K12 a -GAL. Whether or not these effects might be due to increased stabil ity of the a -GAL is unclear from their data. Schmid and Schmitt (1976) reported that 2mM dithioerythritol improved the stabil ity of the a -GAL of E. coli coded for by RAF-plasmid D1021. 15 B. o-GAL AND INVERTASE SYSTEMS OF SACCHAROMYCES CARLSBERGENSIS De la Fuente and Sols (1962) were the first to present convincing evidence that the invertase and o-GAL systems of S. carlsbergensis were distinctly different than the catabolic pathways of other sugars such as maltose and lactose. 0-Fructosides and o-galactosides are hydrolzyed outside the cell membrane while other disaccharides are transported into the cell by specific permease prior to hydrolysis, thus placing o-GAL and invertase in a small group of yeast exoenyzmes which includes acid phosphatase, glucanases, and esterase (Lazo et al., 1977). Three of these enzymes (acid phosphatase, invertase, and o-GAL ) have been shown to be glycoproteins (Neumann and Lampen, 1967; Boer and Steyn-Parve, 1966; Lazo et al., 1978), and it has been suggested that the carbohydrate may be involved in the secretory process (Lazo et al., 1977). In the case of invertase early studies indicated that two forms of this enzyme were present in Saccharomyces yeasts. The first, called the internal or light form, was released only by rupture of the cell wall membrane, had a molecular weight of 270,000 and lacked a carbohydrate moiety. The external, or heavy form was released by formation of protoplasts, had a molecular weight of 135,000, and contained 50% mannan and 3% glycosamine (Gascon et al., 1968). The kinetic parameters and molecular weights of proteins of the light and heavy forms were very similar, but some differences in amino acid contents were noted. Gascon et al., (1968) concluded that the two forms probably had one or more common subunits which were the active portions of the enzymes. In 1975, Moreno et al. reported the presence of multiple forms of invertase which were intermediate in size between the heavy and light forms previously reported. All intermediate forms (of which three were 16 detected) were exclusively intracellular. It is interesting to note that when the yeast was grown in conditions which induced the invertase system, the heavy invertase predominated, while in yeasts where invertase was repressed, the various internal forms accounted for the majority of the activity. The authors suggest that the lighter forms are in some sense precursors of the external enzyme and may in fact represent sequential addition of carbohydrate fragments to a common peptide moiety. The exoenzyme acid phosphatase system of 5. cerevisiae has also been studied in an attempt to clarify the role of glycosylation in a yeast enzyme secretion. Mizunaga and Noguchi (1982) treated yeast protoplast with tunicamycin, an antibiotic which blocks glycosylation but not protein synthesis, and examined the protoplasts for accumulation of a non-glycosylated acid phosphatase precursor. They presented convincing, although not conclusive evidence, that three such precursors were present, but in an inactive form. Lazo et al., (1977; 1978) have reported studies of o-GAL from S. carlsbergensis. These workers grew the yeast in the presenceof galactose (an inducer of o-GAL). They detected only a single form of o-GAL and concluded that no structurally distinctive intracellular o-GAL was present. The o-GAL had a native molecular weight of 300,000 and consisted of 52% mannose, 4% glucose, less than 1% glucosamine, and the remainder protein. SDS-PAGE indicated the presence of two or three subunits with molecular weights between 90,000 and 100,000. These investigators also examined other physical and kinetic properties of this enzyme. The isoelectric point was estimated to be 3.6. With PNPG as a substrate, the pH optimum was approximately 4.5. Enzyme activity remained constant for at least 24 h 17 when held at 30°C in buffers from pH 3 to pH 8. Thermal stabil ity was also excellent, in that the enzyme retained 85% of its original activity after 150 min at 50°C and pH 7.5. Km and Vmax values calculated for 3 different substrates are presented in Table 6. C. FLATULENCE AND LEGUME CONSUMPTION Excessive flatulence has long been associated with the consumption of legumes and legume products. Bacterial origin of flatus was established relatively early in the study of this problem. Intestinal gas production can be virtually eliminated by treatment with various antimicrobial drugs (Bond and Levitt, 1978; Hellendoorn, 1973). At one time the belief that flatus was largely caused by aerophagia (ie. the swal lowing of air) was widely held, but this was refuted by evidence that flatus is composed primarily of H 2, C 0 2 and, in some individuals methane, gases which are not abundant in air. Studies with dogs indicated that most gas is • formed by bacterial fermentation in the ileum and colon (Richards and Steggerda, 1966; Rackis et al., 1970a). Food residues which are not digested by the human are believed to provide a fermentable substrate for resident bacterial gas formers in the lower bowel. In the early sixties it was established that the a-galactoside sugars (ie.melibiose, raff inose, stachyose and verbascose) are not hydrolyzed or absorbed by the mammalian digestive system (Taeufel et al., 1965). However Taeufel et al., (1965) did detect low levels of a-galactosidase activity in human feces, presumably originating from intestinal bacteria. The o-galactoside sugars, particularly stachyose and raff inose are present at quite high levels in most legumes, and are known to be utilized by some bacteria (Fleming, 1981; Ulezlo and Zaprometova, 1982) 18 Table 6. Kinetic properties of purified o-GAL of Saccharomyces carlsbergensis based on data from Lazo et al., (1978). Substrate Km (mM) Vmax (umoles/min/mg protein) PNPG 6 125 Melibiose 6 140 Raffinose 135 100 19 Since Taeufel's group published their results, many studies have been undertaken to conclusively identify the flatulence factors of legumes (Hellendoorn, 1969; Kurtzman and Halbrook, 1970; Murphy et al., 1972; Rackis et al., 1970b; Rockland et al., 1969; Steggerda 1968; Wagner et al., 1976). Murphy et al., (1972) showed that the flatulence potential resided mainly in a 60% ethanol soluble fraction which contained the sugars fructose, sucrose, raffinose, and stachyose, as well as various small peptides. Other studies likewise indicated the involvement of the indigestible sugars. Calloway and Murphy (1968) demonstrated that breath hydrogen (also of intestinal bacterial origin) was measurably increased by consumption of as little as 2g of stachyose. Raffinose also increased breath H2. Several studies have examined bacteria isolated from mammalian feces for the ability to produce gas from a-galactosides. Evidence from these studies indicates that anaerobic spore formers of the genus Clostridium and Clostridium perfringens strains in particular may be the major causitive organisms (Richards et al., 1968; Rackis et al., 1970b; Rockland et al., 1969). Garg et al., (1979) found that five Clostridium sp. isolates all produced gas from legume slurries. The three most productive gas formers were all later identified as C. perfringens. Rockland et al., (1969) found that slurries of various legumes stimulated in vitro growth and gas production of C. perfringens ATCC 3624, but that stachyose and raffinose did not. Sacks and Olson, (1979), however, reported that five different strains of C. perfringens varied greatly in their ability to utilize c-galactosides. None the less, three of five strains utilized raffinose to some extent. There is general agreement therefore that bacterial metabolism of o-galactosides in the colon and ileum contributes to flatulence. On the 20 other hand, there exists a considerable amount of evidence that these sugars are not the only flatulence factors. Beans (Phaseolus vulgaris) contain approximately 4 to 5% a-galactosides, principally stachyose (Fleming, 1981). Calloway (1973)found that consumption of 2 to 4 times as much stachyose or raffinose in pure form was required to ellict the flatus volumes associated with equivalent o-galactoside consumption in the form of cooked beans. Supporting this finding are the results of Murphy et al., (1972) who found that C0 2 egestion as flatus was elevated much less by pure sugars than by equivalent amounts of o-galactosides in cooked beans . Other facts also suggest that the problem is more complicated than was originally supposed. Navy beans contain less a-galactosides than soy beans, but produce twice the volume of intestinal gas (Hellendoorn, 1973). Calloway et al., (1971) demonstrated that the treatment of California small white bean slurry with Diastase 80 (a commercial enzyme preparation with a-galactosidase activity) did not greatly decrease the flatulence potential even though negligable amounts of raffinose or stachyose remained in the slurry. Unfortunately, these investigators did not examine the Diastase 80 treated legumes for melibiose content. Diastase 80 has invertase activity and the hydrolysis of raffinose by invertase yields melibiose and fructose. Without information on melibiose levels the significance of these results cannot be properly evaluated. Finally, Wagner et al. (1976) found a marked synergism between a-galactosides and a-galactoside-free bean residues on gas production in the rat They found that although stachyose and raffinose did elevate flatus levels, the effect was much greater when bean residue was fed simultaneously. The bean component responsible for this effect has yet to 21 be identified. D. THE APPLICATION OF MICROBIAL a-GAL TO FOOD PROCESSING Two major uses for a-GAL within the food industry have been suggested: 1. treatment of soy milk to remove indigestible a-galactoside sugars: 2. treatment of beet molasses to remove raffinose. Both of these applications have been studied in laboratory or larger scale experiments and have shown promise of success if used on an industrial scale. 1. a-GAL TREATMENT OF SOY MILK Sugimoto and Van Buren (1970) first suggested that consumer acceptance of soy milk might be enhanced if the a-galactoside flatulence factors stachyose and raffinose were removed. They demonstrated hydrolysis of these sugars when soluble a-GAL from Aspergi I lus saitoi was added directly to soy milk at pH 6.2 and 50°C. Sufficient enzyme was employed to ensure virtual removal of a-galactosides within three hours. Thananunkul et al., (1976) treated soy milk with crude a-GAL isolated from Mortierella vinecea and immobilized in polyacrylamide gel granules. The granules were packed into a continuous flow fluidized bed- apparatus. Although hydrolysis as high as 60% could be achieved at low flow rates, the authors concluded that the procedure was too slow to be practical and that an immobilized enzyme with improved stability and activity was required. Cruz et al. (1981) used Cladiosporium cladiosporoides a-GAL for direct application to soy milk, and reported virtual removal of oligosaccharides in six hours. 22 The use of hollow-fibre membrane technology has also been examined to some extent. Smiley et al. (1976) used an AMICON DC-30 hollow fibre dialyzer to hydrolyze stachyose and raffinose in soy milk. A crude a-GAL preparation from AspergiIlus awamori was held inside the hollow filters, while soy milk or whey was recirculated through the jacket surrounding the fibers. The system has three advantages over batch addition of enzyme. First, the enyzme is conserved and may be recovered. Secondly, because soy proteins can not pass through the membrane of the hollow fibers, proteolysis is avoided, even if active proteolytic enzymes are present in" the a-GAL preparation. Thus crude a-GAL preparations can be utilized. Finally, no non-dializable constituents of the enzyme preparation remain in the product after treatment. The studies mentioned above have established that enzymatic removal of a-galactoside from soy milk is feasible. No doubt further research and development would lead to more efficient and inexpensive immobilized a-GAL treatments. Apart from the technical aspects of the problem two fundamental questions remain. Does a-GAL treatment significantly decrease the flatulence potential of soy milk and given that it does, would a-GAL treated soy milk find greater consumer acceptance than the untreated product? These questions must be answered before the process can be properly evaluated. 2. o-GAL TREATMENT OF SUGAR BEET MOLASSES Raffinose is found in sugar beet at levels of up to approximately 0.15%, particularily after extended storage (Suzuki et al., 23 1969). It is extracted in the beet juice and is progressively concentrated as sucrose is crystallized out of the molasses. Raffinose finally constitutes 6-10% of the beet molasses, at which levels it inhibits normal sucrose crystallization. The molasses must then be discarded. Removal of the raffinose allows better sucrose yields and decreases the problem of molasses disposal. The use of o-GAL for this purpose was first proposed by Suzuki and Tanabe, (1963). This group later isolated the enzyme of Mortierella vinecea and successfully demonstrated the process (Suzuki et al., 1969). The o-GAL of this fungus has a pH optimum of 4.5 and to accommadate this, the beet molasses was adjusted to pH 5.2. A lower pH was not acceptable because acid inversion of sucrose occured at pH's below 5.0. They also noted the need to eliminate invertase activity from enzyme preparations. Apparently the study by Suzuki et al., (1969) is the only published scientific paper on the treatment of beet molasses with o-GAL. Several patents, however, have been granted for processes to produce microbial o-GAL, and to treat beet juice and beet molasses (Narita et al., 1975; Suzuki et al., 1972; Watanabe et al., 1974). E. DETERMINATION OF INITIAL VELOCITIES. KM AND VMAX OF ENZYME  CATALYZED REACTIONS 1. INITIAL VELOCITY The initial velocity (V0) of an enzyme substrate reaction is progressively reduced as substrate is depleted and products accumulate such that product inhibition begins. Allison and Purich (1979) es t imated that the initial cond i t ion typical ly persisted for 10 to several hundred s e c o n d s depending upon the enzyme, the substrate concentrat ion, the buf fer ing capac i ty of the medium and the ratio of enzyme to substrate concentra t ions . In theory, the initial ve loc i ty is the react ion ve loc i ty at t ime zero. The most c o m m o n procedure for est imating V„ is to measure the react ion rate at as l ow an e n z y m e concentrat ion as is pract ical , and over as short a t ime per iod as is consistent with proper mixing of the reactants, reso lu t ion l imi tat ions of the assay method, etc. Even so , there is no guarantee that product inhibition or substrate dep let ion will not s ign i f i cant ly a f fec t the est imate (A l l i son and Purich, 1979). A decep t i ve picture m a y emerge when the initial rate per iod is short, in which V, 's at l ow substrate concentrat ions appear to be lower than they actual ly are. Th is is because initial rate per iods are shorter at l ow substrate concentrat ions than at high substrate concentrat ions , due to substrate deplet ion. If a f ixed time a s s a y per iod is c h o s e n wh ich is with in the initial rate per iod at high substrate concentrat ion but b e y o n d the initial per iod at low concentra t ion , the M i c h a e l i s - M e n t o n plot of ve loc i ty vs initial substrate concentrat ion wi l l be s i g m o i d a l rather than hyperbol ic (A l l i son and Purich, 1979). 2. K M A N D V M A X Enzyme ca ta lyzed react ions wh ich fo l low M i c h a e l i s - M e n t o n k inet ics can be d e s c r i b e d by the f o l l o w i n g equation. V. = (2) 25 where V 0 is the initial ve loc i t y , S the substrate concentrat ion , Vmax the maximum obtainable v e l o c i t y , and Km the M i c h a e l i s - M e n t o n constant . This equation desc r ibes a rectangular hyperbo la which passes through the origin and approaches a hor izontal asympto te as V , approaches Vmax. Km c o r r e s p o n d s to the substrate concentrat ion at which V, equals 1/2 Vmax. Unfor tunate ly the hyperbo la does not lend itself to easy graphic interpretat ion. C o n s e q u e n t l y , b iochemists have for years e m p l o y e d var ious rearranged f o r m s of the M i c h a e l i s - M e n t o n equat ion wh ich result in linear graphic representat ions of the react ion . The m o s t famil iar f o r m is the double inverse plot of L ineweaver and Burke (1934). Km and Vmax can be eas i l y ca lcu la ted f rom the s l o p e and intercept of the result ing line. Graphic ana lys is of this type is s imp le , quick, and may prov ide adequate accuracy for s o m e purposes (C le land, 1967). The methods however , have t w o important s h o r t c o m i n g s . In the first p l a c e , graphic ana lys is d o e s not p r o v i d e any numer ica l measure of the rel iabi l i ty of the f i t ted c o n s t a n t s . Such i n fo rmat ion may be essent ia l for proper eva luat ion o f the exper imental resul ts in relation to theoret ical cons iderat ions and fo r c o m p a r i s o n wi th the results of other invest igators (Wi lk inson, 1961). The second s h o r t c o m i n g of graphic determinat ions of Km and Vmax concerns the nature of the exper imental error of V„ measurements . {In most instances substrate concent ra t ions are known with suff ic ient accuracy that they do not contr ibute mater ia l ly to (3) 26 experimental error. For practical purposes therefore, experimental error of Km and Vmax estimates may be considered in terms of initial velocity measurements. (Cleland, 1979).} In graphic analysis there is an intrinsic assumption that the variances of each initial velocity determination are uniform and that each experimental point is of equal value in estimating the line and ultimately the kinetic parameters. In fact, the limited experimental data which is available on this point indicate that the assumption is unwarranted. Storer et al. (1975) found "no support for the generalization that (initial) velocities should be homogeneous in variance"; rather, variance appeared to be proportional to the true velocity. Askelof et al., (1976) suggested that experimental points be weighted by the inverse of experimentally determined variance or, alternately by 1/V0 where V 0 equals initial velocity and x is a constant determined experimentally for each enzyme. Oestreicher and Pinto, (1983) recommended that the weighting factor 1/V02 be employed, when experimental estimates of variance at each substrate level were not available. Cleland, (1979) made a similar recommendation for the general case but argued that as long as velocities did not vary by more than a factor of 5, the assumption of constant variance was reasonable. Statistical non-linear regression methods which allow estimates of standard error of Km and Vmax have long been available (Wilkinson, 1961; Cleland, 1967). These methods also make inclusion of weighting factors convenient. The fact that these methods have not been more widely used can probably be ascribed to the extensive and complex calculations required. Even when made 27 available in Fortran for computer analysis (Cleland, 1967) the methods were not easily accessable to all. Recently the programs have been rewritten in Basic for use with moderately priced pocket computers (Oestreicher and Pinto, 1983). In this form the non-linear regression calculations are not only more satisfactory from a theoretical point of view, they are also faster than graphic analysis. F. SPORULATION OF CLOSTRIDIUM PERFRINGENS Spores of C. perfringens are notoriously difficult to obtain in labatory media (Sacks and Thompson, 1978). The induction of sporulation of this species has been studied by many investigators, both as part of basic research into the mechanisms of sporulation and, in recent years in relation to enterotoxin formation (Kim et al., 1967; Fredien and Duncan, 1973; Labbe and Rey, 1979; Sacks, 1982). Enterotoxin, the causative agent of C. perfringens food borne illness, is exclusively associated with sporulation. Fredien and Duncan (1973) suggested that enterotoxin is in fact a particular spore coat structural protein. Thus studies of the enterotoxin require an efficient and reliable means of inducing sporulation of C. perfringens. To date, a large number of sporulation media have been suggested for this organism (Angelotti et al., 1962; Duncan and Strong, 1968; Elner, 1956; Gyobu and Kodama, 1976; Nishida et al., 1969). All of these media have been shown to be effective in some cases. Problems arise primarily because the nutrient requirements for sporulation vary greatly between strains (Muhammed et al., 1975). Also, because the mechanism of endospore induction is not yet understood, there are few guidelines available to assist in the development of a universally satisfactory C. perfringens medium (Sacks and Thompson, 1977). 28 The inhibitory effects of large amounts of readily utilizable carbohydrate on sporulation are generally recognized. This may be due to the anti-sporulation effects of bacterial metabolites such as aldehydes, oxaloacetic acid, and lactic acid or to some unknown factor (Muhammed et al., 1975). The stimulation of sporulation by certain non-nutrient substances has been reported by Sacks and Thompson, (1977) and Sacks, (1982). Several methylxanthines, described as purine analogs, increased spore yields of some C. perfringens strains, as does the vasodilator drug papaverine. The mechanism of action is not clear. Duncan-Strong medium is probably the most widely used sporulation medium for C. perfringens, a fact which may be attributed to the ease of. preparation, low cost, and high success rate of the preparation (Labbe and Rey, 1979; Duncan and Strong, 1968). In this case starch is employed as the carbohydrate source. Starch is only slowly utilized by most C. perfringens strains and presumably for this reason it is less inhibitory than some other carbohydrates. However several papers have reported that spore yields from some strains of C. perfringens are greater when raffinose is substituted for starch (Labbe and Duncan 1977; Labbe et al., 1976; Labbe and Rey, 1979). Labbe and Rey (1979) also showed that enterotoxin production increased with spore counts when raffinose was used and suggested that this modified medium might be particularily suitable for enterotoxin studies. II. PURIFICATION AND CHARACTERIZATION OF THE o-GALACTOSIDASE OF CLOSTRIDIUM PERFRINGENS A. INTRODUCTION Legumes are widely consumed as low cost sources of dietary protein. Excessive flatulence however, long associated with ' legume consumption, may limit the acceptability of legumes in the diet. Several studies have shown that the c-galactoside sugars raffinose and stachyose contribute to flatulence (Murphy et al., 1972; Rackis et al., 1970; Steggerda et al., 1966). These sugars are not hydrolyzed or absorbed by the mammalian digestive system (Taeufel et al., 1965). The sugars therefore pass into the lower bowel, where they may be fermented by various resident bacteria, with the production of gas. Strains of Clostridium perfringens in particular have been implicated as major sources of intestinal gas (Richards et al., 1968; Sacks and Olson,. 1979). Some studies have suggested that other factors besides c-galactosides may contribute to flatulence (Calloway, 1973; Calloway et al., 1971; Wagner et al., 1976). An a-galactosidase similiar or identical to the bacterial enzyme present in the large intestine could be a valuable research tool for clarifying the role of these sugars in flatulence. The objective of this study was to isolate and characterize the a-galactosidase of C. perfringens (a-GALCp). B. MATERIALS AND METHODS 29 30 1. MICROORGANISMS Twenty strains of C. perfringens, isolated from fecal and non-fecal sources were donated by the Division of Laboratories, British Columbia Ministry of Health. One other strain was isolated from a soil sample. All were identified by the method of Hauschild, 1975. Strain M34 was confirmed to be C. perfringens by means of fhe API Anaerobe identification system (Analytab Products, Plainview, N.Y.). Strains were maintained as spore suspensions in distilled water at 4°C. Spores were induced and counted by the methods of Labbe and Rey (1979), employing either raffinose or starch as the sporulation carbohydrate. Short term culture maintainence utilized Cooked Meat medium (Difco). Strains capable of producing acid and gas within 36 h at 45°C from 2.0% trypticase peptone (BBL), 0.35% agar, 0.002% phenol red and 0.5% carbohydrate were considered positive for that carbohydrate. For production of o-GALCp, cultures were grown at 42°C in o-Galactoside Broth (1.5% trypticase peptone, 0.5% proteose peptone, 0.5% sodium chloride, 0.2% dipotassium phosphate, and 0.1% sodium thioglycollate, with or without 0.5% raffinose and 0.5% melibiose). The fermentation procedure was as follows. Spore suspension was heat shocked (75°C; 20 min) and surface plated onto Germination Agar (1.5% trypticase peptone, 1.0% yeast extract, 1.5% agar). Germination plates were incubated overnight at 42°C. Single colonies were inoculated into Cooked Meat medium, incubated 24 h, and a loopful of culture used to inoculate 15 mL of o-Galactoside Broth (with or without c-galactosides). This culture was incubated overnight, then 31 used as the inoculum for 125 mL of o-Galactoside Broth, which in turn, after 6 h incubation, was the inoculum for 2,500 mL of o-Galactoside Broth. Following germination of the Spores, no special steps were taken to ensure anaerobiosis, except that freshly autoclaved media was used throughout and cultures were grown without shaking or mixing. 2. PURIFICATION OF o-GALCP Cells harvested by centrifugation (9,000 x g; 4°C), washed with 3 volumes of buffer, (0.05M monopotassium phosphate, pH 6.7) and resuspended in the same buffer, were disrupted by passage thorough a cold Aminco-French Pressure Cell (Silver Spring, Md), at 15,000 psi and 2°C. The crude extract was clarified by centrifugation (27,000 x g; 30 min; 4°C). a. DEAE Cellulose Chromatography Crude extract (100-500 units of a-GALCp) was applied to a 2.5 cm x 22 cm column of Whatman DE-32 DEAE cellulose (Whatman Chemical Seperation Ltd., England), previously equilibrated with 0.02M monopotassium phoshate buffer, pH 6.7. The column was washed with 300 mL of starting buffer and the sample was eluted with a 0.02 -0.05M sodium chloride gradient (total volume, 840 mL). Active fractions were pooled and concentrated by ultrafiltration at 4°C in an Amincon (Lexington, Mass.) Model 52 cell fitted with a Diaflo PM-10 membrane. 32 b. Gel Fi l trat ion Chromatography Gel f i l trat ion was carr ied out at 2°C in a 2.6 cm x 92 cm co lumn of Sephacry l S400 (Pharmacia, Inc., Dorva l , Que.), equi l ibrated with 0.05M potass ium phosphate buf fer , pH 6.7. Fract ions (6 mL) were co l l ec ted and a s s a y e d for o - g a l a c t o s i d a s e act iv i ty and absorbance at 280 nm. Molecu lar weight es t imat ions were made by c o m p a r i s o n with protein standards catalase (liver), bovine serum albumin (BSA) , and o v a l b u m i n , all suppl ied by S i g m a (St. Louis , MO) . Ca lcu lat ions were as descr ibed by Frei fe lder (1976). 3. E N Z Y M E A C T I V I T Y o - G a l a c t o s i d a s e act iv i ty w a s moni tored by means of a f ixed t ime (15 min) assay at 45°C. The react ion mixture conta ined 0.67mM p-n i t ropheny l a l p h a - D - g a l a c t o p y r a n o s i d e (PNPG), 0.06M sod ium phosphate buffer (pH 6.5), 1.5 m g B S A / m L , 3 m M cys te ine , and an appropriate amount of e n z y m e . The react ion w a s s topped by the addit ion of 5 v o l u m e s of 0.1 M sod ium carbonate and the absorbance read at 405 n m . One unit of enzyme was def ined as the amount required to h y d r o l y s e 1 umole of P N P G per min . The absorbance of 1 ug p-n i t ropheno l/mL was 0.124. 4. E L E C T R O P H O R E S I S S o d i u m dodecy l sulphate po lyac ry lamide gel e lec t rophores is ( S D S - P A G E ) was carried out in a 12 cm x 12 cm vert ical slab e lec t rophores i s unit (Atta Inc., Japan) by the method of Laemml i (1970). Protein bands were loca ted , by means of a C o o m a s s i e blue stain cons i s t ing of 27% i s o p r o p a n o l , 10% acetic ac id , and 0.04% 33 Coomassie brilliant blue R-250 (BioRad, Richmond, CA) in water. Gels were stained overnight, then destained with 12% isopropanol and 7% acetic acid in water. Calculation of corresponding molecular weights was by the method described by Weber and Osborn, (1969). 5. ISOELECTRIC FOCUSING Analytical horizontal polyacrylamide gel isoelectric focusing (IEF-PAGE) was carried out in a Bio-Rad Model 1415 electrophoresis cell, according to the manufacturers instructions. Gel slabs were 45 mm x 125 mm and either 0.8 or 1.6 mm thick. Bands were located by means of a Coomassie blue protein stain and/or by a PNPG a-galactosidase activity stain in which the gel was placed on filter paper and flooded with the solutions described in the enzyme assay .Following incubation the reaction was stopped by flooding the gel with sodium carbonate solution. The pH gradient was measured with a surface pH electrode (0.5 cm probe; Corning Sci. Products, Medfield, MA) and a Fisher Accumet pH meter, Model 620. 6. THERMAL STABILITY Duplicate 0.1 mL samples of purified enzyme solution were incubated for 15 min at various temperatures in 12 mm x 100 mm test- tubes. Test tubes were pre-equilibrated in water baths at the test temperatures and experiments were staggered to ensure that incubation times were equivalent to within 2 sec. The enzyme was then immediately assayed against PNPG at 35°C. Relative activity was determined by comparing activity to that of enzyme incubated at 2°C for a correspnding period of time and assayed against PNPG at 34 35°C. 7. SUBSTRATE AFFINITY Fixed time assays (1 min) were used to estimate initial velocities of enzyme substrate reactions (pH 6.5; 40°C; enzyme concentration 8.0 ug protein/mL) at 6 PNPG concentrations ranging from 0.12 to 3.0 mM. Km, Vmax, and standard errors for each were computed using the non-linear regression program of Oestreicher and Pinto, 1983. 8. PROTEIN DETERMINATIONS Protein determinations were done by the method of Lowry et al (1951) as modified by Peterson (1977). Crystalline BSA (Sigma) was used as a standard. C. RESULTS AND DISCUSSION 1. STRAIN SELECTION The ability of Clostridium perfringens to produce acid and gas from fructose, sucrose, raffinose, and melibiose is illustrated in Table 7. Of the 21 strains examined, 2 strains demonstrated rapid utilization of both raffinose and melibiose. One of these, designated M34, was used for further study of the o-GALCp. 2. SPORULATION Sporulation of C. perfringens is frequently difficult to induce and many complex media have been developed for this purpose. Of 35 Table 7. Production of acid and gas from carbohydrates by strains of C. perfringens . Substrate Strain Fructose Sucrose Raffinose Melibiose F1 . + + + -F2 + - - -F4 + + - -F5 + + - -F7 + + - -FO + + + + FA + + + -M06 + + - -M20 + + + -M21 + + + -M22 + + + -M30 + + - -M31 + + - -M34 + + + + M40 + + - -M64 + . + - + M74 + + - -M75 + + -M81 + + + -M92 + + - + M24FS + + + -+ = Acid and gas produced : - = no acid or gas produced. 36 these the medium of Duncan and Strong (1968) has gained the widest acceptance. Labbe and Rey (1979) reported that replacement of the starch in Duncan Strong medium with raffinose improved spore recoveries in 6 out of 8 C. perfringens strains tested. In this study 12 of 21 strains gave higher spore counts with raffinose (Table 8). Neither carbohydrate, however, was preferred by all strains and one strain failed to sporulate in either medium. 3. YIELDS OF o-GALACTOSIDASE When C. perfringens M34 was grown in broth containing 0.5 % raffinose and 0.5 % melibiose, the yield of a-GALCp in the crude cell extract was 1.2 U/g washed cells (wet weight). Cell free growth medium contained little or no a-GALCp activity. When the strain was grown without exposure to a-galactosides, a-GALCp in the crude cell extract was only 0.07 U/mL. Apparently, in this strain least, a-GALCp is constituitive but partially inducable. C. perfringens is known for its ability to grow rapidly at 45°C and results presented here indicate that strain M34 was capable of utilizing a-galactosides at that temperature. The reason 42°C was chosen for production of the enzyme was the possibility that genetic control of o-GALCp might be plasmid mediated. Whether this is the case in C. perfringens has not been determined, but it has been shown to be the case for an a-galactosidase of Escherichia coli K12 (Schmid and Schmitt, 1976). C. perfringens has been shown to harbour a variety of plasmids (Duncan et al., 1978). Growth at 46°C has been suggested as a way of "curing" C. perfringens of plasmids (Rood et al., 1978). In light of the possibility of plasmid control of the 37 Table 8. Spore counts of C. perfringens strains grown in Duncan Strong sporulation medium with starch or raffinose as the carbon source (thousands of spores / ml) Strain DS with Starch DS with Raffinose FO 320 12.1 F1 400 a N.D. F2 140 1450 F4 40 6.7 F5 52 1580 F7 510 230 FA 6200 2400 M06 169 170 M20 10 160 M21 a N.D. 24 M22 330 1900 M30 0.02 365 M31 2.7 5.4 M34 800 98 M40 0.18 244 M64 a N.D. 16 M74 a N.D. a N.D. M75 250 0.02 M81 2600 2.2 M92 a N.D. 1800 M24R 0.80 82 a N.D.= none detected. 38 enzyme, it was thought prudent to use a lower temperature to grow the organism. 4. PURIFICATION OF o-GALCP The results of the purification procedure are summarized in Table 9. Cell free crude extract, applied to a DEAE cellulose column and eluted with a linear sodium chloride gradient gave a single peak of c-GALCp activity at a salt concentration of 0.19 M (Figure 1). The active fractions were pooled, concentrated by ultrafiltration, and applied to a Sephacryl S400 column. Active fractions were again pooled, concentrated, and reapplied to the same column. The pooled activity peak from this column represents the highest degree of purity acheived. SDS-PAGE of this fraction indicated the presence of one major band and at least two weaker protein bands (Figure 2). Further attempts to improve purity were abandoned because of poor recovery of activity. Attempts to chromatograph a-GALCp on Sephadex G150 at room temperature resulted in loss of >60% of the activity. A final attempt to improve purity by reapplying the sample to DEAE cellulose resulted in a loss of greater than 90% of the enzyme activity. Therefore further characterization was carried out with the Sephacryl S400(2) active fractions. 5. PH OPTIMUM Relative rate of hydrolysis of PNPG was greatest at pH 6.3 (Figure 3). The pH optimum of a-GALCp in the crude extract was similiar. 39 Table 9. Effects of the purification steps on specific activity and yield of o-GALCp. Procedure Activity (U/mL) Specific Activity (U/mg protein) Yield (%) French Press. DEAE Cellulose Sephacryl S400 Sephacryl S400 (2) 1.21 0.72 0.18 0.13 0.17 0.81 1.61 100 64 50 32 a - G a l a c t o s i d a s e A c t i v i t y , (U/mL) OQ c ft 1 fl> OQ P h-> • P o rt n O 3 " to H- o o. 3 P P c/> rt CD **\ cw P o P rt ^ 3 " < X rt o P. / \ 1 C D > > n to o o 3 O c t n 3 > m o 3 * o o t—• »—• c o . t—' ct> o to o CD o 3 O P (5 cr 3 t/> rt o H •i P cr rt P M - 3 o O 3 CD o < 0 re A b s o r b a n c e , (280 nm) < i o ON Sodium C h l o r i d e , (M) 4 1 Figure 2. SDS-polyacrylamide gel e l e c t r o p h o r e s i s of o(-GALCp and p r o t e i n standards; A, oC-GALCp peak from Sephacryl S400 chromatography; B, c a t a l a s e ; C, bovine serum albumin (polymerized). 42 F i g u r e 3. pH optimum o f ot-GALCp a t 30"C. Each p o i n t r e p r e s e n t s t h e a v e r a g e o f d u p l i c a t e d e t e r m i n a t i o n s . 43 6. M O L E C U L A R W E I G H T S The native molecular weight of o - G A L C p , as est imated by means of gel f i l trat ion on Sephacry l S400, was approx imate ly 97,000 (Figure 4). A l though it was not poss ib le to pos i t i ve l y identi fy the enzyme band on S D S - P A G E , the most prominent band cor responded to a molecular weight of 46,000 (Figure 5). On this bas is , the native protein appears to be a d imer . 7. ISOELECTRIC POINT The enzyme was f o c u s e d on a pH 4.0 to pH 6.5 gel gradient and located by means of an act iv i ty stain, at pH 5.6± 0.1. No other band of act iv i ty was noted . 8. AFFINITY FOR PNPG Km and Vmax, plus or minus their standard errors were computed by the program of Oestre icher and Pinto (1983) to be; Km = 0.20 ± 0.02 m M ; Vmax = 2.02 ± 0.06 uM/min . A L ineweaver Burk plot of the same data is included to a l low a visual est imate of g o o d n e s s of fit (Figure 6). Linear regress ion of the L ineweaver Burk plot resulted in the f o l l o w i n g parameter va lues : Km = 0.24 m M ; Vmax = 2.16 uM/min. 9. A C T I V A T I O N OF o - G A L C P BY V A R I O U S C O M P O U N D S L o s s of enzyme act iv i ty was a recurrent p rob lem throughout the iso lat ion procedure. Gel f i l t rat ion at r o o m temperature typ ica l ly resulted in loss of at least 60%. Chromatography at 2°C improved reco ve r i e s . The microbia l inhibitor sod ium azide (0.02 %) and to a 44 Figure 4. Gel f i l t r a t i o n chromatography of molecular weight standards on Sephacryl oc-GALCp and S400 . F i g u r e 5 . w e i g h t E l e c t . r o p h o r e t i c m o b i l i t y o f tf-GALCp and m o l e c u l a r s t a n d a r d s on SDS - p o l y a c r y l a m i d e g e l . -5.0 0 5.0 10.0 1/S F i g u r e 6. L i n e w e « v e r Burk p l o t o f o(-GALCp k i n e t i c d a t a o f PNPG h y d r o l y s i s a t pH 6.5 and 40*C: V - v e l o c i t y (umoles/ m i n ) ; S « s u b s t r a t e c o n c e n t r a t i o n (mM). 47 lesser extent chlorhexidine gluconate (0.002 %) also decreased activity (Table 10). In assays at 45°C, 2-mercaptoethanol, NADH, cysteine, and BSA each increased the apparent activity of GALCp. The combination of BSA and cysteine had the greatest effect and was used in routine assays. In an attempt to elucidate the mechanism of activity enhancement by these compounds, their effect on activity at 30°C was examined. At this lower temperature no activation was noted. Other authors have noted the activation effect of 2-mercaptoethanol and NADH on the a-galactosidase of E. coli K12 (Burstein and Kepes, 1971). The effect of BSA was examined because it had been described as improving the heat stability of the a-galactosidase of Bacillus stearothermophilus (Pederson and Goodman, 1980). It would appear that in the case of a-GALCp the reducing environment and/or the presence of the protein in the form of BSA, serve to increase the apparent activity of the enzyme. None the less, the question remains as to whether the reducing environment alters the active site of the enzyme, or whether it serves to stabilize the enzyme against thermal denaturation. If the former was the case one would expect activity to be increased at all temperatures, while in the latter case one would expect to see an effect only at temperatures sufficiently high to initiate thermal denaturation. Since the reducing compounds did not increase apparent activity at 30°C but did at 45°C, it is likely that the mechanism of activation is improvement of thermal stability. 48 Table 10. Effects of various compounds on a-GALCp activity at 30°C and 45°C. Compound Cone. (mM) Relative Activity (%)* 30°C 45°C sodium azide 3.0 76 chlorhexidine gluconate 0.03 89 BSA 0.02 103 144 NADH 1.3 97 120 cysteine 3.0 102 114 mercaptoethanol 100 96 122 BSA-NADH 0.02,1.3 156 BSA-cysteine 0.02,3.0 163 BSA-mercaptoethanol 0.02,100 151 * 100 % Relative activity was activity at a given temperature in assay buffer without additives. 49 10. HEAT STABILITY Heat stability of a-GALCp decreased markedly as purity increased. The enzyme in the crude extract had an apparent temperature optimum of 47°C in a 15 min assay (Figure 7). The purified enzyme however, was 96% inactivated by 15 min at 45°C (Figure 8). This fact very likely accounts for at least some of the apparent enzyme losses during the purification procedure, as the routine assay temperature during purification was 45°C. 50 100 -75 -50 • 25 -• — I 1 1 1 1 1 , i i 22 27 32 37 42 47 52 57 Temperature ( c ) g u r e 7. T e m p e r a t u r e optimums o f cx-GALCp i n c r u d e e x t r a c t ( T ) and i n a c t i v e f r a c t i o n s o f S e p h a c r y l S400 c h r o m a t o g r a p h y ( • ) . Each p o i n t r e p r e s e n t s t h e a v e r a g e o f d u p l i c a t e d e t e r m i n a t i o n s . 10 20 30 40 50 T e m p e r a t u r e (*C) u r e 8. T h e r n a l s t a b i l i t y o f oC-GALCp, i n c u b a t e d at v a r i o u s t e m p e r a t u r e s f o r 15 min, t h e n a s s a y e d at 35°C. 52 D. CONCLUSIONS This study has reported the partial purification and characterization of c-galactosidase from C. perfringens. The enzyme has an apparent molecular weight of 96,000, an isoelectric point of pH 5.6, and a pH optimum of 6.3. Km was 0.20 mM raff inose. Precise information on this enzyme may be useful in elucidating the mechanism of gas production in vivo . A l so the enzyme itself could prove to be a valuable research tool for in vitro and animal studies of the flatulence potential of legume components. The C. perfringens c-galactosidase, however, would be unsuitable for the treatment of human foods because of the pathogenic nature of C. perfringens. III. ISOLATION AND CHARACTERIZATION OF THE o-GALACTOSIDASE OF WASTE LAGER YEAST (SACCH AROMYCE S CARLS BE RGE NS/S). A. INTRODUCTION a-Galactosidase treatment has been suggested as a means of removing the oligosaccharides raffinose and stachyose from legumes and legume products such as soybean milk (Delente et al., 1974; Smiley et al., 1976; Sugimoto and van Buren, 1970; Cruz et al., 1981). A similiar enzyme treatment can be used to remove raffinose from sugar beet molasses, thereby improving the crystallization yields of sucrose (Delente et al., 1974; Suzuki et al., 1969). For either application an enzyme source which is inexpensive and toxiologically unimpeachable is a important prerequisite. In this study it was found that waste lager yeast had relatively high levels of a-galactosidase activity. Waste brewers yeast is in plentiful supply and probably represents the cheapest source of this enzyme currently available. Furthermore, brewers yeast is known to be non-pathogenic and is an acceptable source of another enzyme which is approved for use in some food processes on Canada (i.e. lactase) (Anon., 1981). An c-galactosidase from this species of yeast has been previously isolated and described (Lazo et al., 1977, 1978). Isolation of a-galactosidase from waste lager yeast has not been described. The objective of this study was to isolate and characterize the a-galactosidase from waste brewers yeast (a-GALSc). . 53 54 B. MATERIALS AND METHODS 1. BREWERS YEAST Waste lager yeast was donated by Molsons Brewery, Vancouver, B.C. The yeast was identified by Molsons to be a strain of Saccharomyces carlsbergensis . 2. CRUDE EXTRACT Lager yeast was washed with 3 volumes of 0.05M TRIS-HCI buffer, pH 7.2, and resuspended in the same buffer at 10% total solids. Cells were ruptured by passage thorough a cold (2°C) French pressure cell at 15,000 psi, and clarified by centrifugation (27,000 x g; 30 min; 4°C) 3. ISOPROPANOL PRECIPITATION Crude extract was mixed with isopropanol (-20°C) in a ratio of 1 to 1.6 volumes, immediately filtered thorough a pad of Celite '545' (Fisher Sci., Pittsburgh, PA) (6% by weight of crude extract), then resuspended in 0.75 volumes of 0.5M TRIS-HCL buffer and stirred (4°C, 10 hours). Celite was removed by filtration. 4. SEPHADEX G100 GEL FILTRATION CHROMATOGRAPHY of Isopropanol extract (20 mL) was applied to a 2.5 cm x 70 cm column of Sephadex G100 (fine) (Pharmacia Inc., Dorval, Quebec) held at 2°C. A flow rate of 35 mL/h was used. Fractions (6 mL) were collected and a-galactosidase active fractions pooled. 55 5. ANION EXCHANGE CHROMATOGRAPHY A 2.5 cm x 22 cm column of Whattman DEAE cellulose DE-32 (Whatman Chemical Seperation Ltd., England) was equilibrated with Mcllvaine buffer (0.017M citric acid and Na2HP04 to pH 5.6). Pooled fractions from the Sephadex G100 column (100-300 units) were applied to the DEAE cellulose column, washed with 300 mL of starting buffer, and then eluted with a linear 0-0.20M sodium chloride gradient (dissolved in the same buffer) totalling 700 mL. 6. SEPHACRYL S400 GEL FILTRATION CHROMATOGRAPHY Active peaks from the DEAE cellulose chromatography were concentrated by ultrafiltration at 4°C, in an Amincon (Lexington, MA) Model 52 cell fitted with a Diaflo PM-10 membrane, at 4°C, and applied to a 2.6 x 92 cm column of Sephacryl S400 held at 2°C and equilibrated with 0.05M KH2P04-NaOH buffer (pH 6.7). 7. PROTEIN MEASUREMENTS Protein determinations were by the method of Lowry et al., (1951), as modified by Peterson (I977). Crystalline bovine serum albumin (BSA) (Sigma, St. Louis, MO.) was used as a standard. 8. o-GALACTOSIDASE ACTIVITY Activity was assayed by means of a 15 min fixed time assay in duplicate, at pH 4.5 and 42°C. The assay mixture contained 0.3 mL of 2mM p-nitrophenyl c-D-galactopyranoside (PNPG), 0.6 mL of buffer (Mcllvaine, 0.1M citric acid), and 0.1 mL of appropriately dilute enzyme. The reaction was stopped by the addition of 5 mL of 0.1M 56 sodium carbonate and the absorbance at 405 nm determined. The absorbance of1 ug p-nitrophenol/mL was 0.124. One unit of activity is defined as sufficient enzyme to hydrolyze 1 umole PNPG/min under these conditions. Activity against raffinose was assayed by determining released galactose with a lactose/galactose enzymatic analysis kit (Boehringer Mannheim, Dorval, Quebec). 9. MOLECULAR WEIGHT ESTIMATION Molecular weights were determined by gel filtration as outlined by Andrews (1965), using the Sephacryl S400 column described above. Monomer molecular weights were estimated by sodiun dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was carried out in a 12 x12 cm vertical slab electrophoresis unit (Atto Inc., Japan) by the method of Laemmeli (1970). Protein bands were located by means of a stain consisting of 27% isopropanol, 10% acetic acid, and 0.04% Coomassie brilliant blue R-250 (BioRad, Richmond, CA). Gels were stained overnight, then destained with 12% isopropanol and 7% acetic acid. Calculations of corresponding molecular weights was as described by Weber and Osborn, (1969). Molecular weight standards (ovalbumin, catalase, r-globulin, and BSA) were obtained from Sigma. Polymerized BSA, in which monomer and dimer BSA bands were easily detected, was prepared with glutaraldehyde by the method of Payne, (1973). 57 10. ISOELECTRIC F O C U S I N G Ana ly t i ca l p o l y a c r y l a m i d e gel isoelectr ic f o c u s i n g ( I E F - P A G E ) was done in a BioRad M o d e l 1415 e lec t rophores is ce l l , accord ing to the manufacturers instruct ions. Gel s labs were 45 m m x 125 m m and either 0.8 m m or 1.6 m m thick. B io ly te ampho ly tes (BioRad, R i chmond , C A ) with a pH 3.7 to pH 6.2 gradient were used. The gradient w a s measured with a sur face pH probe (0.5 cm diameter probe; Corn ing S c i . Products , M e d f i e l d , M A ) and a Fisher A c c u m e t pH meter , M o d e l 620. The o - ga lac tos idase band w a s located by f l o o d i n g the f resh ly f o c u s e d gel with the so lut ions descr ibed in the e n z y m e assay . Fo l l owing incubat ion, the react ion was s topped by f l ood ing the gel with 1M sod ium carbonate. Regions of o - G A L S c act iv i ty were indicated by a y e l l o w co lour . 11. S U B S T R A T E AFFINITY Initial react ion ve loc i t i es at var ious substrate concentrat ions were es t imated by two di f ferent procedures .Enzyme concentrat ion was 0.1 ug protein/mL react ion mixture. In the f irst instance, f ixed t ime a s s a y s (20 min with P N P G ; 30 min with r a f f i n o s e ) were used at 40°C with V o taken as the average ve loc i ty over that per iod . In the s e c o n d instance, V o was est imated f rom the first der ivat ive of the substrate enzyme react ion curve at t ime zero . The react ion was carr ied out asept ica l ly in s toppered glass serum bott les . S a m p l e s were taken with steri le syr inges through the s toppers , analyzed for the react ion product and a substrate deplet ion curve constructed . This was a c c o m p l i s h e d by f irst determining the react ion order which best fit the data at each substrate concentrat ion by the opt imizat ion of 58 data transformation for linearization technique of Fujii and Nakai (1980). Equations were then fitted to the transformed data by linear regression. In both cases, values for Km, Vmax, and their corresponding standard errors were computed with the program of Oestreicher and Pinto (1983) for fitting the Michaelis-Menton equation to experimental data by means of non-linear regression analysis. 12. DETERMINATION OF PH OPTIMUM The effect of pH on activity was examined using PNPG (0.67mM) and raffinose (20mM) as substrates, in Mcllvaine's buffer (0.17M citric acid, 0.2M Na2P04, pH 3.5 to 6.5). Activity was determined as described in the enzyme assay. Activities were expressed as percentages of the activity at pH 4.5. C. RESULTS AND DISCUSSION 1. YIELDS One gram (dry wt.) of washed, waste lager yeast yielded 27 units of a-GALSc in the clarified crude extract fraction. The cell wall fraction was not assayed for o-GALSc. 2. PURIFICATION The results of the purification procedures are summarized in Table 11. The isopropanol precipitation step provided a rapid, convenient, and economical first purification step, which might easily be scaled up for handling larger batches. It did, however, result in 59 Table 11. Purification of o-GALSC by 2-Propanol precipitation, G100 Sephadex gel filtration, DEAE cellulose and Sephacryl S400 gel filtration chromatography. Procedure Activity Protein Specific Yield Activity U/mL mg/mL U/mg (%) Protein Crude Extract 2-Propanol Prec G100 Sephadex DEAE Cellulose (total) DEAE (peak A) (S400 peak A) DEAE (peak B) (S400 peak B) DEAE (peak C) (S400 peak C) 1.79 1.18 1.11 0.10 0.38 0.54 0.53 0.46 0.40 16.08 2.98 0.18 0.007 0.011 0.004 0.11 0.40 6.17 54.8 48.2 88.8 100 70 56 45 11 15 19 60 approximately 30% loss of enzyme activity. Chromatography on Sephadex G100 resulted in a mixed peak of a-GALSc and other proteins just following the void volume of the column. Approximately 80% of the activity was recovered. Elution of the a-GALSc from DEAE cellulose resulted in three active peaks. (Figure 9). The presence of three peaks in this chromotogram was unexpected. Lazo et al., (1977) reported only one peak of a-GALSc activity from S. carlsbergensis when chromatographed on DEAE Sephadex A-50. Consequently an effort was made to determine whether other differences between the peaks could be detected. The peaks were collected separately, concentrated by ultrafiltration and applied separately to a Sephacryl S400 column. When compared to molecular weight standards, a-GALSc-A appeared to have a molecular weight of 118,000, a-GALSc-B a molecular weight of 95,000, and a-GALSc-C a molecular weight of 65,000 (Figure 10). BSA, catalase, ovalbumin, and y-globulin were used as molecular weight standards. r-Globulin behaved somewhat anomalously, a characteristic of some glycoproteins which has been well documented (Andrews, 1965). a-GALSc enzymes are also glycoproteins (Lazo, 1977), and the possibility that this affected the gel filtration charactistics can not be excluded. 3. SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL  ELECTROPHORESIS a-GALSc peaks were also distinctive in terms of their SDS-PAGE profiles (Figure 11). A diffuse, poorly resolved protein band corresponding to molecular weights of approximately 65,000 to 62 u r e 10. G e l f i l t r a t i o n o f <x-GALSc-A, ac-GALSc-B, a(-GALSc-C and p r o t e i n s t a n d a r d s on S e p h a c r y l S400. 63 gure 11. SDS-polyacrylamide gel e l e c t r o p h o r e s i s of a(-GALSc f r a c t i o n s and bovine serum albumin: A, crude e x t r a c t ; B, ol-GALSc peak from Sephadex G100 column; C, <*-GALSc-A from DEAE c e l l u l o s e column; D, cX-GALSc-B from DEAE c e l l u l o s e column; E, ct-GALSc-C from DEAE c e l l u l o s e column; F, bovine serum albumin (polymerized). 64 130,000 can be seen to be progressively . intensified by the purification process. DEAE cellulose peaks A, B and C can be seen to correspond to large, intermediate, and small molecular weight segments of the larger band, a result generally consistent with their apparent molecular weights as determined by gel filtration. 4. ISOELECTRIC FOCUSING a-GALSc peaks A, B and C were focused by IEF-PAGE and the active bands detected by means of a PNPG activity stain. All three showed single bands of activity at pH 4.4 + 0.1. 5. MULTIPLE MOLECULAR FORMS OF a-GALSC Lazo et al. (1977) reported only one form of a-GALSc in 5. carlsbergensis, a large extracellular glycoprotein with a molecular weight of 300,000. This enzyme was collected from cultures grown in a galactose containing medium designed to induce a-GALSc. A structurally distinct internal enzyme was not found. In this study, involving o-GALSc isolated from cell free extracts of waste lager yeast, external enzyme was not detected, but multiple, lower weight forms (65,000 - 130,000) of the enzyme were detected. The most extensively studied exocellular enzyme of Saccharomyces is invertase (Lazo et al., 1977). Invertase has been suggested as a model for the study of glycoprotein synthesis and secretion by yeasts (Moreno et al., 1975; Gascon et al., 1968). In the case of invertase, multiple molecular forms have been detected. The external enzyme is apparently homogeneous, with a molecular weight 65 of approximately 270,000, while at least three smaller internal forms have been reported (Moreno et al., 1975). Moreno et al. (1975) suggested that the internal forms of invertase represent sequential addition of carbohydrate units to a common polypeptide. Other authors have suggested that the multiple forms may be degradation products and that only two true forms exist; an internal protein and a external glycoprotein (Lazo et al., 1977). The relative proportions of internal and external invertase have been shown to depend upon the conditions under which the yeast was cultured. The larger, external form predominates when the invertase system is induced. (Sutton and Lampen, 1962). The information presented here suggests that the a-GALSc system may be comparable to the invertase system. The apparent discrepancies between the results of Lazo et al, (1977) and the study reported here may be due to differences in culture conditions. In the fomer study, the external form was favoured, while in this study the internal form predominated. Futhermore, the extraction procedure used here, (French press disruption of washed cells) was not suitable for the recovery of external enzymes. Further study is required to determine, first whether an external enzyme can be recovered from waste lager yeast and, secondly, whether differences can be detected in the carbohydrate moiety of the external and internal enzymes. Also the possibility of artifacts due to degradation products requires clarification. 66 6. PH OPTIMUM The relationship of the activity of o-GALSc-C to pH was examined (Figure 12). a-Galactosidase activity towards PNPG and raffinose was maximum at pH 4.5. Lazo et al. (1977) reported the same pH optimum for the a-GAL they isolated from S. carlsbergensis, using PNPG as a substrate. Similar results were obtained with the crude extract of the present study. The shape of the a-galactosidase-pH curves for PNPG and raffinose were different even though the optimum pH was the same. The activity towards raffinose was somewhat higher than towards PNPG at pHs above the optimum. Since a-GALSc showed substantial activity towards raffinose at pH 5 to 5.5, it might find use in the sugar processing industry where more acidic conditions must be avoided to prevent acid inversion of sucrose (Suzuki et al., 1969). 7. SUBSTRATE AFFINITY Km and Vmax values were determined for a-GALSc-C at 40°C and pH 4.5, for both PNPG and raffinose (Table 12). Initial velocity (Vo) was first estimated at different substrate concentrations by fixed time assays. This method of determining Vo requires an assumption of linearity in the reaction curve up to the point of the assay, an assumption that is frequently difficult to justify theoretically or validate experimentally (Allison and Purich, 1979). In practical terms, the assumption of linearity may decrease the precision of the Km and Vmax calculations. In an attempt to circumvent the problem, an alternate approach was used to calculate Vo in which Vo values were 67 ure 12. The e f f e c t o f pH on a c t i v i t y o f a-GALSc-C w i t h PNPG ( • ) and r a f f i n o s e ( O ) as s u b s t r a t e s . Each p o i n t r e p r e s e n t s t h e a v e r a g e o f d u p l i c a t e d e t e r m i n a t i o n s . Table 12. Kinetic parameters of a -GALSc (± standard errors) and residual standard errors (RSE), computed with the program of Oestreicher and Pinto (1983) using initial velocities estimated by fixed time assays or derived from experimentally determined curves. Substrate Parameter Vo by Fixed Derived Vo Time Assay PNPG Km (mM) 2.54 ±0.32 2.58 ±0.11 Vmax (uM/min) 21.4 ± 1.4 23.8 ±0.5 RSE (uM/min) 3.10 x 10-1 1.13 x 10-' Raffinose Km (mM) 25.7 ±2.1 16.7 ±1.2 Vmax (uM/min) 43.9 ±1.9 41.5 ±1.3 RSE (uM/min) 4.32 x 10-1 4.26 x 10 1 69 derived from experimentally determined enzyme reaction curves (Figure 13). The equations which best fit the experimental data, together with the calculated Vo values are presented in Table 13. Km and Vmax values were computed for each set of Vo determinations by the method of Oestreicher and Pinto (1983). This method was preferred to the usual graphic analysis for two reasons. The Lineweaver Burke Plot, although emminently convenient, can be deceptive because of the inherent assumption that all points in the plot are of equal weight, when in fact it has been shown that varience varies directly with reaction velocity (Storer et al., 1975; Askelof et al., 1976; Nimmo and Mabood, 1979). The Oestreicher Pinto (1983) procedure employed weighting factors ( inverse velocity squared) to compensate for this. Secondly, the Lineweaver Burke procedure provided no estimate of the reliability of the fitted constant. The Oestreicher Pinto program, in addition to Km and Vmax values, provided standard errors of each parameter and the residual standard error. The two methods for determing Vo gave comparable parameter values for each substrate. Parameter standard errors and residual standard errors were decreased by the curve fitting method. 70 Time ( h r ) F i g u r e 1 3 . H y d r o l y s i s o f PNPG b y d - G A L S c - C a t 4 0 " C , pH 4 . 5 , a n d 5 d i f f e r e n t i n i t i a l PNPG c o n c e n t r a t i o n s . 71 Table 13. Results of curve fitting to o-GALSC kinetic data at various initial substrate concentrations. Substrate Initial Regression Equation R 2 PNPG Raffinose Cone. (mM) 0.24 0.48 1.00 2.00 4.00 4.00 6.67 20.0 40.0 Derived Vo (umoles/min) Iny - -0.84x - 1.18 0.9997 2.01 y-(M = 0.42x + 1.34 1.0000 3.77 y - l = 0.65x + 1.00 0.9999 6.50 y-tt7 = 0.23x + 0.62 0.9997 10.56 y - l = 0.09x + 0.25 0.9999 14.46 Iny = -0.20x + 1.39 0.9932 8.2 Iny = -0.18X + 1.90 0.9932 12.0 Iny = -0.11x + 3.00 0.9967 22.1 y - l = 0.0018X + 0.025 0.9993 29.5 72 D. CONCLUSIONS The waste lager yeast supplied by Molson's Brewery proved to be an abundant source of a-galactosidase, yielding 27 U of activity/g (dry wt.) of washed yeast. The low cost, availability and proven safety of brewers yeast make it an ideal commercial enzyme source. This study has demonstrated that a-GALSc can be recovered by a rapid cell disruption-solvent precipitation procedure. Further purification, if desired can be achieved by gel filtration and/or DEAE cellulose ion exchange chromotography. 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