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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  STUDIES OF a-GALACTOSIDASES PERFRINGENS  ISOLATED FROM  AND WASTE LAGER YEAST  CLOSTRIDIUM  (SACCHAROMYCES  CARLSBERGENSIS)  by TIMOTHY DOUGLAS DURANCE A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  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  c  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 agree  shall that  make  it  permission  freely for  available  extensive  for  reference  copying  of  this  and  study.  thesis  I further  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  purification and characterization of a Clostridium  1 describes  perfringens  o-galactosidase  while part 2 describes the isolation and characterization of from  waste  lager  yeast  (Saccharomyces  carlsbergensis,  the partial  a-galactosidase  also  known  as S.  uvarum).  Of  21 strains of C. perfringens  melibiose  while  tested, 10 utilized either raffinose or  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 was  of the enzyme at 45°C decreased as purity  only  partially  reversed  by the addition  of  increased. This  2-mercaptoethanol,  trend 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  molecular  cellulose weights  of  chromatography. the  Subsequent  a-galactosidase  gel  in peaks  filtration  A, B  118,000, 95,000, and 65,000 respectively. The isoelectric  indicated  and C  point  of  to be  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 o f Tables  vi  List of Figures  .V i i i  Acknowledgements  x  I. Literature Review A.  1  Microbial o-Galactosidases  1  1.  Distribution  1  2.  Localization Cells  of  c-GAL  in  Prokaryotic  3.  Isolation and Purification  4  4.  Physical Properties  5  5.  Kinetic Properties of o-GALs  '.  o-GAL and Invertase systems of Saccharomyces  C.  Flatulence and Legume Consumption  D.  The Application of Microbial  F.  Eukaryotic 1  B.  E.  and  o-GAL  8 15  carlsbergensis  17 to Food Processing  .21  1.  o-GAL Treatment of Soy Milk  21  2.  c-GAL Treatment of Sugar Beet Molasses  22  DETERMINATION OF INITIAL VELOCITIES. Km AND Vmax OF ENZYME CATALYZED REACTIONS  23  1.  Initial Velocity  23  2.  Km and Vmax  24  Sporulation of  II. Purification Clostridium  and  Clostridium  perfringens  Characterization  of  27  the  perfringens  c-Galactosidase  of 29  A.  Introduction  29  B.  Materials and Methods  29  1.  Microorganisms  ^.  2.  Purification of o-GALCp i i i  30 31  C.  3.  Enzyme  4.  Electrophoresis  32  5.  Isoelectric  33  6.  Thermal  7.  Substrate  8.  Protein  Results  32  Focusing  Stability  33  Affinity  34  Determinations  and  34  Discussion  1.  Strain  2.  Sporulation  3.  Yields  4.  Purification  5.  pH  6.  Molecular  7.  Isoelectric  8.  Affinity  9.  Activation  34  Selection  34 34  of  a-Galactosidase of  36  o-GALCp  38  Optimum  10. Heat D.  Activity  38  Weights  43  Point  for  43  PNPG  of  43  o-GALCp  by  Various  Compounds  .43  Stability  49  Conclusions  III. I s o l a t i o n Lager  and  Yeast  52 Characterization  (Saccharomyces  A.  Introduction  B.  Materials  of  the  a-Galactosidase  of  Waste  carlsbergensis)  53 53  and  Methods  1.  Brewers  2.  Crude  3.  Isopropanol  4.  Sephadex  5.  Anion  .'.  Yeast  54  Extract  54  Precipitation  G100  Exchange  54  Gel  Filtration  54 Chromatography  Chromatography i v  54 55  6.  Sephacryl  S400  7.  Protein  8.  o-Galactosidase  9.  Molecular  10.  Isoelectric  IV.  55  and  55 [  55  Estimation  56  Focusing  57  Affinity of  57 pH  Optimum  58  Discussion  58  1.  Yields  58  2.  Purification  58  3.  D.  Chromatography  Activity  Weight  12. D e t e r m i n a t i o n Results  Filtration  Measurements  11. S u b s t r a t e  C.  Gel  Sodium  Dodecyl  Sulfate  Polyacrylamide  Gel  Electrophoresis  60  4.  Isoelectric  64  5.  Multiple  6.  pH  7.  Substrate  Focusing  Molecular  Forms  Optimum  of  c-GALSc  64 „  66  Affinity  66  Conclusions  ~  References  ~  v  ;  72 73  List of Tables  Table  Page  1.  Microbial sources of a-GAL  (Ulezlo and Zaprometova, 1982)  2.  Microbial  have  o-GAL's  which  been  studied  in  2  highly  purified  preparations 3.  Physical  6  properties  of  some  microbial  o-GAL's;  molecular  weights  (MW) and isoelectric points (pi) 4.  Substrate  specificity  of  7 from  o-GAL  Vic/a  aglycon  faba:  specificity  (Dey and Pridham, 1972)  9  5.  Kinetic properties of some microbial o-GAL's  6.  Kinetic  properties  of  purified  o-GAL  of  11  Saccharomyces  carlsbergensis  based on data from Lazo et al., (1978) 7.  Production  of  acid  and gas  from  18  carbohydrates  by  strains  of C.  perfringens  8.  Spore  35  counts  sporulation  of  C.  medium  strains  perfringens  with  starch  or  grown  raffinose  in  Duncan  Strong  as the carbon  source  (thousands of spores/mL) 9.  Effects  of  purification  37 steps  on  specific  activity  and  Effects  of various  compounds  on o-GALCp  activity  at 30°C and  45°C 11.  48  Purification gel  of  filtration,  o-GALSc DEAE  by  2-propanol  cellulose  and  precipitation, G100 Sephadex  Sephacryl  S400  gel  chromatography 12.  of 39  o-GALCp 10.  yield  Kinetic  parameters  filtration 59  of  c-GALSc  (±  standard  errors)  and  residual  standard errors (RSE), computed with the program of Oestreicher and Vi  Pinto (1983) using initial velocities estimated by fixed time assays or derived from experimentally determined curves 13.  Results  of  curve  fitting  to  a-GALSc  substrate concentrations  kinetic  68 data at  various  initial 71  v i i  List of Figures  Figure 1.  p Chromatography  of  o-GALCp  on  DEAE  cellulose:  a g e  absorbance. (•);  a-galactosidase activity, ( A ) ; sodium chloride concentration. (.  .)  40  2.  SDS-polyacrylamide  gel  electrophoresis  of  and' protein  a-GALCp  standards; A, a-GALCp peak from Sephacryl S400 chromatography; B, catalase; C, bovine serum albumin (polymerized) 3.  pH optimum  of  a-GALCp  at 30°C. Each  point  41 presents  the average  of duplicate determinations 4.  Gel  filtration  42  chromatography  of  a-GALCp  and  molecular  weight  standards on Sephacryl S400 5.  Electrophoretic  mobility  of  44 a-GALCp  and molecular  weight  standards  on SDS- polyacrylamide gel 6.  45  Lineweaver Burk plot of a-GALCp kinetic data of PNPG hydrolysis at pH 6.5 and 40°C: V =velocity 0  (uM/min); S  = substrate concentration  (mM) 7.  46  Temperature active  optimums  fractions  of  of  in  a-GALCp  Sephacryl  S400  crude  extract  chromatography  (•)  (•). Each  represents the average of duplicate determinations 8.  Thermal  stability  of  a-GALCp.  incubated at various temperatures for 51  DEAE cellulose chromatography  of a-GALSc:  (•);  chloride  absorbance.  (•); sodium  a-galactosidase  gradient,  (••-). A,  Gel  filtration  of  o-GALSc-A,  a-GALSc-B,  viii  activity,  B, and C  indicate areas of a-galactosidase activity. 10.  point 50  15 min, then assayed at 35°C 9.  and in  61 a-GALSc-C  and  protein  standards on Sephacryl S400 SDS-polyacrylamide bovine  serum  Sephadex D,  gel  albumin:  62  electrophoresis A,  crude  extract;  G100 column; C, o-GALSc-A  o-GALSc-B  from  DEAE  of  cellulose  o-GALSc B,  fractions  o-GALSc  peak  and from  from DEAE cellulose column; column;  E,  a-GALSc-C  from  DEAE cellulose column; F, bovine serum albumin (polymerized) The  effect  raffinose  of (o)  pH as  on  activity  substrates.  of Each  duplicate determinations Hydrolysis of  PNPG by o-GALSc-C  initial PNPG concentrations  o-GALSc-C point  with  represents  PNPG the  63 (•)  averge  and of 67  at 40°C, pH 4.5, and 5 different 70  Acknowledgements I wish to express his  encouragement,  study. I would W.D.  Powrie,  also and  my  gratitude to my supervisor, Dr. B.J. Skura, for  guidance,  and  like to  thank  Dr.  P.M.  assistance  throughout  the  course  of  this  my graduate committee, Dr. S. Nakai, Dr.  Townsley  assistance.  x  for  their  valuable  suggestions  and  I. LITERATURE REVIEW  MICROBIAL  g-GALACTOSlPASES  1. DISTRIBUTION, The  earliest  o-galactosidases back  to  references  in  the  scientific  galactohydrolases  (o-D-galactoside  1895 when Bau and Fisher and Lindner  literature  to  (E.C. date  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  filamentous  time  fungi  numerous  have  been  bacteria, actinomycetes, yeasts, and 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 bacteria  question  and  fungi  of  the  cellular  has  received  location  some  of  a-GAL  attention  in  because  both  of  the  bearing location has on purification techniques and on the mechanism of  a-galactoside metabolism. In general, the bacterial  to be intracellular. In the case of the Escherichia encoded enzyme sugars  a-GAL, is  active  Schmid  and  Schmitt  within  the  cytoplasmic  are transported  hydrolysis. Akiba  and  into the cell Horikoshi  1  (1976)  coli  (I978) examined  that  the  a-Galactoside  permeases the  appear  D1021 plasmid  determined  membrane.  by specific  a-GALs  prior  possibility  to that  Table  1. Microbial  Bacteria  and  A erabacter  Bacillus  B.  B.  sources  Actinomycetes  aenogenes  ceneus  deltrukii  steanotherm ophilus  of  a-GAL  (Ulezlo  and  Zaprometova,  Filamentous  A ^xrgillus  1982).  Fungi  Yeasts  C andida  niger  guillerm ondii  A.  fum igatus  Pichia  gjillierm ondii  A.  jeanseim ei  Saccharom ycetes  A.  niger  S.  cereviaae  hiepenas  carlsbergensis  Bacteriodes  fragilis  A.  orysae  S.  C lostridium  perfringens  A.  rubenscens  S.  A.  saiatoi  Schisosaccharom  terreus  S.  malidevoraus  S.  octoaiorus  C.  m aebashi  0 iplococcus  pneum onia  A.  Escherichia  coli  A.  L actobaa'llus  M icrococcus  Pseudom onas  Salmonella  spp.  spp.  atlantica  typhimurium  4.  terricda  wentu  acremonlum  muscae  W ortierella  vinacea  canescens  Streptococcus  bovis  Penidllium  Streptom yces  fnadiae  P.  var.  Schwanniomyces  Cephalosporium  Circinella  italicus  davforme  melibiosi  yces  japonicus  alluvius  oiivaoeus  P.  rces'gpinus  P.  dupontii  P.  frequentans  cydcpium  P.  janth  P.  paxSUus  P.  piacarum  P.  thorn ii  4  the  a-GAL  of  a  Micrococcus  periplasmic  space  by  freeze-thaw  treatments  strain  attempting and  might  to  with  be  release  lysozyme  located  the  in  the  enzyme  treatment,  with  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 Pseudomonas  atlantica  indicated  that  the  active  enzyme  of was  intracellular, although an inactve form of the enzyme was detected in the periplasmic of  the  space (Day et al., I975). In Bacteroides  a-GAL  activity  could  activity  was  associated  be recovered from  the  with  cell  free  the  fragi/is,  cells  most  but  supernatant  some  (Berg et  al., 1980). The possibility of periplasmic location was not examined. In the of  a-GAL enzyme  fungi  can  Mortierella niger  limited  number of  appear  be  to  recovered  vinacea  (Suzuki  cases which have  been  at  extracellular. The  be from et  least the  partially  mycelium  al., 1969). The  is secreted from the mycelia  free  a-GAL  in a similiar  studied the  filtrates  of  fashion  of  Aspergillus (Adya and  Elbein, 1977). At  least  one  extracellular  in  carlsbergensis  (Martinez  enzyme,  however,  form  the  can  of  a-GAL  yeasts et  been  Saccharomyces  al.,  also  has  1982 be  ;  Lazo  found  shown  cerevisiae et  within  al., the  to  be  and  S.  1977).  Active  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  standard enzyme ammonium  in  Table  purification  sulfate  2.  These  studies  methods, including  precipitation,  DEAE  have  shown  solvent  cellulose  that  precipitation,  ion  exchange  chromatography, and gel filtration can be successfully employed with a-GAL. Recently, affinity  chromatography  for  o-GAL  rapid  purification  Sweeley,  1973).  of  The  succinoylaminoalkyi  (Harpaz  adsorbents  agarose  in  et  al., 1974  used  were  conjunction  amine  o-D-galactopyranosyl  has been successfully used  and  ; Mapes  and  Sepharose  or  with  the  ligands  p-aminophenyl-melibioside  respectively.  4. PHYSICAL  PROPERTIES  o-GALs physical  from different  properties  (Table  microbial  sources  3). Molecular  weights  from 45,000 for the a-GAL of Aspergillus of  a-GAL  has been  Pennici 11 ium  duponti.  demonstrated, both  Although isoelectric points  vary greatly  in bacteria  have been recorded  niger  The presence  in their  of  to 500,000 for the polymeric  and fungi, in some  structure cases.  (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 a-GALs  have been confirmed of  Aspergillus  carlsbergensis al.,  1977).  glycoproteins.  to be glycoproteins; these  niger,  Mortierel la  vinacea,  and  include the Saccharomyces  (Adya and Elbein, 1977 ; Suzuki et al., 1970 ; Lazo et No  bacterial  a-GALs  have  been  reported  to  be  Table  2. Microbial  Organism  which  have  been  studied  in highly  purified preparations.  Reference  E scherichia  Bacillus  a-GAL's  coli  Schmid  stearotherm ophitlus  A spergillus  niger  Saccharom  rolfai  yces  caridtergens's  Schmitt, (1976).  Pederson  and  Bahl  Agrawal,  Adya  C crticum  and  and  and  Kaji  and  Lazo  et  Goodman,  Elbein,  (1980).  (1969).  (1977).  Yoshihara, (1972).  al., (1977).  Table  3. Physical  properties  Organism  Bacillus  S.  of some  microbial  Monomer  Native  MW  pi  weights (MW) and isoelectric  points (pi).  Reference  312,000  Akiba  81,000  280,000  Pederson  84,000  325,000  sp  stearotherw ophillus  MW  a-GALs; molecular  and Horikoshi,  and Goodman,  125,000  6.2  Berg  329,000  5.1  Schmid  and Schmitt,  200,000  Burstein  and Kepes,  M/cnococoys sp  367,000  Akiba  Aspergillus  45,000  Adya  Bacteroides  fnagilis  £.  coli  D 1 0 2 1 plasmid  £.  coli  K12  82,000  niger  and Horikoshi,  and Elbem,  saitoi  290,000  Sugimoto  A.  auamori  130,000  McGhee  500,000  Arnaud  duponti  Saccharomyces  caiisbergenss  100,000  90,000  300,000  3.6  Lazo  (1980)  et al., ( 1 9 8 0 )  A.  Penicillium  (1976)  (1976)  (1971)  (1976)  (1977)  and van Buren, ( 1 9 7 0 )  et al., ( 1 9 7 8 )  et al., ( 1 9 7 6 )  et al., ( 1 9 7 7 )  8 5. KINETIC PROPERTIES OF A  number  identification functions o-GALs pH  of  of  of  kinetic  the  the  o-GALS properties  enzymes,  enzymes,  for  and  of  understanding  for  assessing  for practical applications. These include  optimum, temperature  are  o-GAL  optimum, and the  important  the  for  physiological  the  suitability  of  substrate specificity,  reaction  parameters  Km  and Vmax. a. Substrate specificity catalyze  a-GALs  the  hydrolysis  of  bonds  o-galactosyl  following the general formula :  ,— »v^\ c-R  ^  4- R'OH  OH  ,  (1)  Y_/-0-R  - f ROH  OH  R'OH is commonly water but both R and R' can be aliphatic or aromatic change  groups. As  in configuration  single carbon of  a-GAL  of  the  first  the  second, the configurations of 3, and 4 must  be similiar  1982). The  carbon  Thus  is  and  a-D-fucosides  are  may be sufficient  activity. Two  substrates;  C6  glycosidases  quite  the hydrogen or hydroxyl  the glycoside  or entirely eliminate in  a rule  factors pyranose  specific. A  group's at any  to severely retard  are particularily ring  must  be  important present;  the H and OH groups at carbons  to  D-galactose  less  significant  0-L-arabinosides  1, 2,  (Ulezlo and Zaprometova, (Dey  and  are  often  Pridham, 1972). 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  specificity  has been extensively of  a-GAL  of  V/c/a  studied faba  is  in some outlined  plant in  a-GALs. The  Table  4.  It  is  Table  4. Substrate  specificity  Substrate  Ethyl  a-D-galactoside  a-D-galactoside  n-Propyl  Phenyl  from  Vida  faba ; aglycon  Vmax  a-D-galactoside  Methyl  of o-GAL  a-O-galactoside  specificity.  (Dey and  (umoles/min/mg protein)  Pridham,  1972).  Km  (mM)  1.66  7.13  1.66  8.93  2.20  6.13  20.30  1.11  o-Cresyl  a-D-galactoside  26.00  m-Cresyl  a-D-galactoside  24.30  1.38  23.00  1.54  1.14  p-Cresyl  a-O-galactoside  o-Nitrophenyl  a-D-galactoside  42.10  m-Nitrophenyl  a-D-galactoside  5.86  p-Nitrophenyl  a-D-galactoside  1.33  10.0  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  suggesting a high affinity  for this  has been noted for many  a-GALs  to  PNPG  is  substrate. High affinity  lowest,  for  PNPG  (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 or  slightly  higher  than  most their  a-GALs Km  for  for melibiose are similiar to PNPG.  The  Michaelis-Menton  constant for raffinose is often considerably higher again than that of melibiose. b. pH Optimum The Table  pH  optimum  5. The  bacterial  of  some  enzymes  neutrality while fungal a-GALs  microbial  tend  to  are  listed  in  active  close  to  a-GALs  be  most  have optimums  in the range of pH 4  to 5. In some instances however, the enzymes may be active over a wide  range  of  Cladiosporium pH  3.8 to  H  ion  concentration.  cladiosporoides pH 6.3  carlsbergensis  retains  (Cruz et  For  example  >80% of  a I., 1981). The  the  maximum activity of  a-GAL  of  a-GAL  from  Saccharomyces  exhibits >80% relative activity from pH 3.8 to 5.2 (Lazo  et al., 1978).  c. Thermal Stability The the  37-40°C  however, studied value  temperature  of  some in  range.  optimum (Ulezlo  noteworthy  detail  because  thermophillic  for  and  most  Zaprometova,  exceptions, of  the  microbial  several  possibility  enzymes. Bacillus  of of  o-GALs  are  in  1982).  There  are,  which  have  been  enhanced  stearothermophiIlus  practical produces  Table  5. Kinetic  properties of some  Organism  E.  coli  pH  K12.  microbial o-GAL.  Optimum  Temperature  Km  Optimum  (mM)  7.5  (' C)  3  10  E.  E.  coli  coll  Bacillus  isozyme  D1021  plasmid  6.8  com m unior  stearotherm  1.  7.2  37  2.  PNPG  Reference  Burstein  &  Kepes,197 1  Mel  0.14  PNPG  3.2  Mel  0.1 1  PNPG  2.32  Mel  0.47  PNPG  Schmid  &  Schmitt,  1976  Kawamura  et al. 1976  Pederson  &  cphilus  73  19  isozyme  Substrate  69  0.53  45  Mel  PNPG  Mel  Goodman.  1980  Micrococcus  X>.  7.5  35  0.47  ONPG  1.5  Mel  12.6  Bacillus  32  6.5  40  A •pergfllus  niger  4-4.5  ONPG  7.9  Mel  0.18  0.50  6.0  A.  awamcri  M ortlenella  5.0  vtnaoea  4.0  50  50  &  Horikoshi,  1976.  Akiba  &  Horikoshi,  1976  Adya  &  Raf  1.0  24.1  Akiba  Raf  PNPG  dadla&oroiOes  5.0  65  1977  et al  1978  Raf  PNPG  30  Mel  36  Raf  0.4  PNPG  0.39  Mel  1.83  Raf  McGhee  Suzuki  Cruz  Cladla&crtum  Elbein.  et a l , 1969. 1970  el a l ,  1981.  Sacchanomyces  carlstxrgenss  4.5  6.0  PNPG  6.0  Mel  135  1. PNPG  =  p-nitrophenyl  Mel  =  melibiose;  Raf  =  raffinose.  a-D-galactosidase;  Raf  Lazo  et al., 1977  14 a-GALs and  which  are stable  Goodman,  cladiosporoides  1980).  for more  than  PeniciIlium  a-GAL's  have  similiar  30 min at 65°C  dupontii  and  stability  (Arnaud  (Pederson  Cladiosporium et  al., 1976;  Cruz et al., 1981). Some  other  a-GALs  unstable. The a - G A L by  Kawamura  incubation  Kepes an  of Escherichia  been  temperatures  apparent  at  of  25°C  low  which  on  the  to  be  coli subsp communior  and  protein  basis  of  remarkably as described  deactivated above.  by  This  concentrations.  (1971) made similiar observations  enzyme  found  et al., (1976) w a s significantly  at  particularily  have  effect  was  Burstein  and  about an E. coli kinetic  data,  15 min  K12 a - G A L ,  appears  to  be  different than the enzyme described by Kawamura et al., (1976). Several  compounds  activity of various 1.5  mg/mL  stability  have  been  a - G A L s . Pederson serum  the  stearothermophi11 us  B.  to  improve  and Goodman  bovine  of  albumin  used  or  gelatin a-GAL.  the  apparent  (1980) found  improved Burstein  that  the and  heat Kepes  (1971) found that N A D , NADH, 2-mercaptoethanol, and crude cell extracts of due  (without  the E. coli  a-GAL  K12 a - G A L . Whether  to increased  Schmid improved  and the  activity) all  stability  of  Schmitt  (1976)  stability  of  R A F - p l a s m i d D1021.  improved  or not these  the a - G A L  the  the apparent  reported a-GAL  effects  is unclear that of  E.  2mM coli  from  free  activity might  their  be  data.  dithioerythritol coded  for  by  15 B. o-GAL AND INVERTASE SYSTEMS De evidence distinctly maltose  la Fuente that the  invertase  different and  and Sols  than  lactose.  (1962) were  and  the  OF SACCHAROMYCES  o-GAL  catabolic  0-Fructosides  the  first  CARLSBERGENSIS  to  present  convincing  systems  of  S. carlsbergensis  pathways  of  other  and  sugars  o-galactosides  are  were  such  as  hydrolzyed  outside the cell membrane while other disaccharides are transported into the cell  by  specific  invertase  in  permease  a  small  prior  group  phosphatase, glucanases, and  to  of  hydrolysis,  yeast  esterase  thus  placing  exoenyzmes  (Lazo  et  which  o-GAL  and  includes  al., 1977). Three  of  acid 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 this  enzyme  internal  were  or  light  membrane, had moiety.  The  of  invertase  present form,  was  a molecular external,  in  or  early  3% glycosamine  molecular  weights  of  indicated that  Saccharomyces released  weight heavy  only  yeasts. The by  of  270,000  form  was  protoplasts, had a molecular weight and  studies  of  rupture and  first, of  lacked  released  two  the a  by  forms  of  called  the  cell  wall  carbohydrate formation  of  135,000, and contained 50% mannan  (Gascon  et  al.,  1968). The  proteins  of  the  light  and  kinetic heavy  parameters  forms  were  and 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 invertase forms  which  previously  were  et al. reported the intermediate  reported. All  in  size  intermediate  presence between forms  of the (of  multiple heavy which  forms  of  and  light  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  repressed,  the  various  activity. The precursors addition  predominated,  authors  of  of  the  while  internal  forms  suggest  external  carbohydrate  in  that  yeasts  accounted  the  lighter  enzyme  and  fragments  to  may a  where  for  an  attempt  to  clarify  the  role  of  majority  are  fact  common  exoenzyme acid phosphatase system of 5. cerevisiae in  the  forms in  invertase  in  was  of  some  represent peptide  the  sense  sequential  moiety.  The  has also been studied  glycosylation  in  a  yeast  enzyme  secretion. Mizunaga tunicamycin,  and  an  synthesis,  (1982)  antibiotic  which  examined  the  and  non-glycosylated although not  Noguchi  acid  blocks  yeast  protoplast  glycosylation  protoplasts  phosphatase  conclusive  treated  for  precursor.  They  but  with  not  protein  accumulation presented  of  a  convincing,  evidence, that three such precursors were  present,  but in an inactive form. Lazo  et  carlsbergensis. (an  These  inducer of  concluded  al., (1977;  that  1978) have  workers  o-GAL). They no  grew  reported  the  detected  structurally  yeast only  distinctive  studies in the  a single  intracellular  of  o-GAL  presenceof form  of  S.  galactose  o-GAL was  o-GAL  from  and  present.  The o-GAL had a native molecular weight of 300,000 and consisted of 52% mannose, 4% glucose, less than SDS-PAGE weights  1% glucosamine, and the remainder protein.  indicated the presence of  between  other physical  90,000  and  two  100,000.  and kinetic properties  of  or three subunits with molecular  These  investigators  this enzyme. The  also  examined  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  stability was  also excellent, in that the enzyme retained 85% of its original activity after 150  min  different  at  50°C  and pH  7.5. Km  Excessive legumes  relatively be  values  calculated  flatulence  and legume  early  has long  been  associated  with  the consumption  products. Bacterial origin of flatus was established  in the study of this  problem. Intestinal gas production can various  antimicrobial  and Levitt, 1978; Hellendoorn, 1973). A t one time the belief  but C0  caused  this 2  by aerophagia  was refuted  and, in some  Studies  with  fermentation et  believed formers  to  by evidence  indicated  in the ileum Food  provide  a  activity  by  Taeufel  flatus  of  which  most  2  by  in air.  bacterial  (Richards  and Steggerda, 1966; Rackis  are not  digested  substrate  for  it was established that  stachyose  mammalian et  sugars,  quite high levels  held,  primarily of H ,  is • formed  by  resident  the human are bacterial  the a - g a l a c t o s i d e  and verbascose)  digestive  system  al., (1965) did detect  low  particularly  in most  stachyose  are not (Taeufel  levels  in human feces, presumably originating from  o-galactoside  that flatus was  air) was widely  is composed  gas  (Bond  gas  bowel.  raffinose, the  that  fermentable  In the early sixties  absorbed  that  and colon  residues  in the lower  (ie.melibiose,  (ie. the s w a l l o w i n g  drugs  individuals methane, gases which are not abundant  dogs  al., 1970a).  However  3  CONSUMPTION  virtually eliminated by treatment with  largely  for  substrates are presented in Table 6.  C. FLATULENCE A N D LEGUME  of  and Vmax  of  sugars  hydrolyzed et  al.,  or  1965).  a-galactosidase  intestinal bacteria. The  and raffinose  are  present  at  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  likewise  indicated the involvement  Murphy  (1968)  bacterial  origin) was  demonstrated  of  that  measurably  small  peptides. Other  studies  the indigestible sugars. Calloway and breath  hydrogen  increased by  (also  consumption  of of  intestinal  as  little as  2g of stachyose. Raffinose also increased breath H . 2  feces  Several  studies  for  ability  these  the  studies  Clostridium major  to  indicates  that  Clostridium  causitive  organisms  et  examined  produce  and  Rockland isolates  have  al., 1969). Garg  gas  bacteria from  anaerobic  perfringens (Richards et  all produced gas from  strains et  et  stimulated in vitro  al.,  (1969)  from  mammalian  a-galactosides. Evidence  spore  al.,  formers in  of  particular  1968;  al., (1979) found  Rackis  that  five  found  that  from  the may  et  al.,  genus be  the  1970b;  Clostridium  legume slurries. The three most  gas formers were all later identified as C. Rockland  isolated  sp.  productive  perfringens. slurries  of  various  growth and gas production of C. perfringens  legumes  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  o-galactosides  general in  the  agreement colon  and  therefore ileum  that  bacterial  contributes  to  metabolism  flatulence.  On  of the  20  other  hand, there  exists  a  considerable  amount  of  evidence  that  these  sugars are not the only flatulence factors. Beans  (Phaseolus principally  a-galactosides,  vulgaris)  contain  approximately  4  stachyose  (Fleming,  1981). Calloway  to  5%  (1973)found  that consumption of 2 to 4 times as much stachyose or raffinose in pure form  was  required to  consumption  o-galactoside finding  are  egestion  ellict  the  as  results  flatus  the  flatus  in the of  was  volumes  form  Murphy  elevated  et  of  associated with  cooked  beans. Supporting  al., (1972) who  much  less  equivalent  by  found  pure  this  that  sugars  than  C0  2  by  equivalent amounts of o-galactosides in cooked beans . Other facts also suggest that the problem was  originally  supposed. Navy  beans, but produce twice Calloway  beans  contain  the volume  of  is more complicated than  less  than soy  a-galactosides  intestinal  gas  et al., (1971) demonstrated that the treatment  (Hellendoorn, 1973). of  California small  white bean slurry with Diastase 80 (a commercial enzyme  preparation with  activity) did  a-galactosidase  not  greatly  even though negligable amounts of slurry. Unfortunately, these treated  legumes  for  and the hydrolysis of Without  information  the  flatulence  potential  raffinose or stachyose remained in the  investigators  melibiose  decrease  did  not  content. Diastase  examine 80  has  the  Diastase 80  invertase  activity  raffinose by invertase yields melibiose and fructose.  on  melibiose  levels  the  significance  of  these  results  cannot be properly evaluated. Finally, a-galactosides  Wagner and  et  al.  (1976) found  a-galactoside-free  a  marked  bean residues  synergism  between  on gas production  in  the rat They found that although stachyose and raffinose did elevate flatus levels,  the  effect  was  much  greater  simultaneously. The bean component  when  bean  residue  responsible for this effect  was  fed  has yet to  21 be identified.  D. THE APPLICATION OF MICROBIAL Two  major  suggested:  uses  for  1. treatment  sugars: 2. treatment  within  a-GAL  of  of  a-GAL TO FOOD  soy  beet  milk  to  the  PROCESSING  food  remove  industry  indigestible  molasses to remove  have  been  a-galactoside  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 acceptance flatulence  and Van Buren (1970) first  of  soy  factors  demonstrated Aspergi I lus  milk  stachyose  hydrolysis  of  saitoi was  50°C. Sufficient  might  be  and  these  added  suggested that  enhanced raffinose  sugars  directly  if were  when  to  the  soy  consumer  a-galactoside  removed.  soluble milk  a-GAL  at  pH  They from  6.2  and  enzyme was employed to ensure virtual removal of  a-galactosides within three hours. Thananunkul isolated gel  from  granules.  et  al., (1976) treated  Mortierella The  vinecea and  granules  were  soy  milk with crude  immobilized  packed  into  a  in  a-GAL  polyacrylamide  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 direct  application  to  soy  oligosaccharides in six hours.  milk,  and  cladiosporoides  reported  virtual  a-GAL for removal  of  22 The use examined DC-30 soy held  to  of  hollow-fibre  some  extent.  membrane  Smiley  et  technology  al. (1976) used  hollow fibre dialyzer to hydrolyze stachyose  milk. A crude inside  the  filters,  while  soy  also  an  been  AMICON  and raffinose in  preparation from AspergiIlus  a-GAL hollow  has  milk  awamori  was  whey  was  or  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  inexpensive aspects  development  immobilized  of  the  would  a-GAL  problem  two  lead  to  more  treatments. Apart fundamental  efficient  from  the  questions  and  technical  remain.  Does  a-GAL treatment significantly decrease the flatulence potential of soy milk  and  greater  given  that  it  does, would  consumer  acceptance  than  must  answered  before  questions  be  a-GAL the the  treated  untreated process  soy  milk  product? can  be  levels  of  find These  properly  evaluated.  2. o-GAL TREATMENT Raffinose  is  OF SUGAR BEET found  in  sugar  MOLASSES beet  at  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  inhibits  normal  6-10% of  sucrose  the beet  molasses, at which  crystallization. The molasses  levels  must  it  then be  discarded. Removal of the raffinose allows better sucrose yields and decreases the problem of molasses disposal. The Suzuki  use of  for this  o-GAL  and Tanabe, (1963). This  Mortierella  vinecea  purpose  group  was first  later  proposed by  isolated the enzyme of  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  occured  pH was not acceptable at pH's below  invertase activity Suzuki  5.0. They  of  also  acid  noted  inversion  of  sucrose  the need to eliminate  from enzyme preparations. Apparently the study by  et al., (1969) is the only  treatment  because  beet  molasses  with  published  scientific  o-GAL. Several  paper  on the  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 CATALYZED  OF  INITIAL  VELOCITIES.  KM AND VMAX  OF ENZYME  REACTIONS  1. INITIAL VELOCITY The  initial  progressively accumulate  velocity  reduced  such  that  as  (V ) of 0  an enzyme  substrate  product  inhibition  is  substrate  depleted  begins.  reaction  and  Allison  is  products  and Purich  (1979) e s t i m a t e d to  several  hundred  concentration, enzyme the  that  to  the  The  reaction  rate  and  over  short  the  Even  as  reactants,  so,  depletion Purich,  is  will  not  1979). A  period to  there  is  be  periods substrate assay  short,  time  time  no  shorter  at  chosen  which  concentration  substrate  AND  but  will  medium the  for  the  initial  10  substrate  and the  ratio  of  velocity  is  concentration consistent  is  emerge  when  substrate This  within  beyond  is  of  sigmoidal  If  rate  initial  plot  proper  mixing  method, or  initial  than  rather  rate high  fixed  time  period  than  rate  at  period  velocity  and  appear  initial  a  etc.  substrate  (Allison  because  initial  the  practical,  concentrations  depletion.  the  is  the  concentrations  substrate  as  inhibition  may  are.  measure  assay  estimate  low  be  the  product  to  with  the  substrate to  of  is  V„  affect  Michaelis-Menton  concentration and P u r i c h ,  at  actually  due  is  that  picture  low  enzyme,  estimating  limitations  V,'s  concentrations,  the  as  significantly  which  for  enzyme  guarantee  are  is  an  period  deceptive  persisted  zero.  procedure low  they  concentration,  at at  vs  high low initial  hyperbolic  1979).  VMAX  Enzyme kinetics  theory,  than  substrate  2. K M  In  lower  period  (Allison  concentrations.  resolution  in  the  the  as  a  upon of  at  at  typically  capacity  common  the  condition  depending  buffering  velocity  most  initial  seconds  substrate  reaction  of  the  catalyzed  c a n be d e s c r i b e d  V. =  reactions by  the  which  following  follow  Michaelis-Menton  equation.  (2)  25  where the  V  is  0  the  maximum  constant. passes  at  which  V,  itself  have  the  equals to  for  easy  inverse  plot  of  Vmax.  the  a  Lineweaver  a  to  various  the  and  most  does  forms  form  not  biochemists of  linear  familiar  as  concentration  Consequently,  in  which  asymptote  hyperbola  result  Burke  hyperbola  substrate the  Vmax  Michaelis-Menton  rearranged  which The  the  horizontal  Unfortunately  reaction.  concentration,  rectangular  interpretation.  equation  of  Km  approaches  employed  Michaelis-Menton  substrate  and  corresponds  graphic  years  representations  and  Km 1/2  the  describes  origin  Vmax.  S  velocity,  equation  through  approaches  velocity,  obtainable  This  V,  lend  initial  is  the  graphic the  double  (1934).  (3)  Km of  and  Vmax  the  can  resulting Graphic  provide  place, the  to  analysis  of  Vmax  this for  two  fitted  the  slope  shortcoming  concerns  the  {In  nature  most  accuracy  purposes  the for  and  intercept  quick,  (Cleland,  shortcomings. any  1967).  The  the  first  measure  information  experimental comparison  may  In  numerical  Such  and  results  with  the  may in  of be  relation  results  of  1961). of of  instances that  simple,  provide  of  and  The  second  some  is  constants.  evaluation  considerations  type  not  (Wilkinson,  sufficient  from  important  investigators  measurements. with  calculated  does  the  proper  theoretical  other  have  analysis  reliability for  of  accuracy  however,  graphic  essential  easily  line.  adequate  methods  be  they  graphic the  determinations experimental  substrate do  not  of  Km  and  error  of  V„  concentrations contribute  are  known  materially  to  26 experimental error. For practical purposes therefore, experimental error of  Km  and Vmax  velocity an  estimates  measurements. (Cleland,  intrinsic  assumption  that  determination  are  uniform  equal  in  estimating  value  parameters. In fact, the on this point al.  may  (1975)  velocities appeared  be  1979).}  the  and  the  and  support  should  be  homogeneous  proportional  for  to  the  the  each  initial  there  initial  ultimately  is  the  data which  is  velocity  point  of  kinetic  is available  is unwarranted. Storer et generalization  in  of  analysis  experimental  indicate that the assumption  be  of  experimental  "no  to  each  line  found  in terms  In graphic  variances  that  limited  considered  variance";  true  that  rather,  velocity.  (initial) variance  Askelof  et  al.,  (1976) suggested that experimental points be weighted by the inverse of V  0  experimentally equals  determined  initial  experimentally  for  recommended  that  experimental available.  variance  velocity each the  estimates  Cleland,  and  of  (1979)  x  enzyme.  weighting  or, alternately is  a  1/V  and be  2  0  a  similar  more than a factor  of  determined Pinto,  5, the assumption of  (1983) when  level were not  recommendation  general case but argued that as long as velocities  where  0  employed,  variance at each substrate made  1/V  constant  Oestreicher factor  by  for  the  did not vary by  constant  variance was  reasonable. Statistical non-linear regression methods which allow estimates of  standard  error  of  Km  (Wilkinson, 1961; Cleland, of not  weighting been  extensive  factors  more and  and  Vmax  1967). These  convenient. The  widely complex  used  can  calculations  have methods  fact  that  probably required.  long also these be  been  available  make  inclusion  methods  ascribed Even  when  to  have the made  27 available in Fortran for computer analysis (Cleland, 1967) the methods were not easily rewritten  accessable  in Basic  to all. Recently the programs have been  for use with  (Oestreicher and Pinto,  priced pocket  computers  1983). In this form the non-linear  regression  calculations are not only  moderately  more satisfactory  from a theoretical  point  of view, they are also faster than graphic analysis.  F. SPORULATION OF CLOSTRIDIUM Spores  of  PERFRINGENS are  C. perfringens  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, food  perfringens  borne  1982). illness,  Enterotoxin,  the  is exclusively  causative  associated  agent  with  of C.  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 for  date, a large  this organism  1956; Gyobu  number of sporulation  (Angelotti  and Kodama,  the nutrient  been suggested  et al., 1962; Duncan and Strong, 1968; Elner,  1976; Nishida et al., 1969). All of these  have been shown to be effective because  media have  requirements  in some cases. Problems for  sporulation  vary  media  arise primarily  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  (Sacks and Thompson, 1977).  satisfactory  C. perfringens  medium  28 The  inhibitory  carbohydrate the  effects  of  large  on sporulation are generally  anti-sporulation  effects  of  amounts  of  readily  recognized. This  bacterial  metabolites  may  such  utilizable be due to  as  aldehydes,  oxaloacetic acid, and lactic acid or to some unknown factor (Muhammed et al., 1975). The stimulation of sporulation by certain non-nutrient been  reported  by  methylxanthines,  Sacks  and Thompson, (1977) and  described  some C. perfringens  as  purine  analogs,  strains, as does  substances has  Sacks, (1982). Several  increased  the vasodilator  spore  yields  of  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  the  carbohydrate  and Strong, 1968). In this source.  Starch  is  only  case  starch  slowly  is employed  utilized  by  most  as C.  strains and presumably for this reason it is less inhibitory than  perfringens  some other carbohydrates. However several papers have reported that spore yields  from  some  substituted  for  and  1979).  Rey,  production suggested  strains  starch  that  this  enterotoxin studies.  (Labbe  Labbe  increased  of  with  and  and Duncan Rey  spore  modified  are greater when raffinose  C. perfringens  (1979) counts  medium  1977; Labbe also when  might  be  is  et al., 1976; Labbe  showed raffinose  that was  particularily  enterotoxin used  and  suitable  for  II. PURIFICATION AND CHARACTERIZATION OF THE o-GALACTOSIDASE  CLOSTRIDIUM  OF  PERFRINGENS  A. INTRODUCTION Legumes protein.  are  Excessive  limit  the  have shown that  contribute to flatulence et  al.,  1966).  mammalian pass  into  resident  digestive  bacteria,  perfringens  the  low  however,  cost  long  acceptability  of  c-galactoside  sugars system  lower  as  sources  associated  legumes  of  dietary  with ' legume  in the  sugars raffinose  diet. Several and stachyose  (Murphy et al., 1972; Rackis et al., 1970; Steggerda  These  the  consumed  flatulence  consumption, may studies  widely  bowel,  with  the  are  not  (Taeufel where  hydrolyzed et  they  production  al.,  1965).  may of  or  be  gas.  absorbed  The  sugars  fermented Strains  by  therefore  by  of  the  various  Clostridium  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  C.  study  perfringens  was  to  isolate  and  characterize  (a-GALCp).  B. MATERIALS AND  METHODS  29  the  a-galactosidase  of  30 1. MICROORGANISMS Twenty non-fecal British  strains  sources  Columbia  of  were Ministry  C. perfringens, isolated donated of  by  the  from  Division  Health. One other  of  strain  fecal  and  Laboratories, 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),  sporulation  employing  carbohydrate.  either  Short  raffinose  term  culture  or  starch  as  maintainence  the  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  o-Galactoside 0.5% sodium  of  o-GALCp, cultures  Broth (1.5% trypticase  were  grown  at 42°C in  peptone, 0.5% proteose  chloride, 0.2% dipotassium  peptone,  phosphate, and 0.1% sodium  thioglycollate, with or without 0.5% raffinose and 0.5% melibiose). The fermentation shocked  procedure  (75°C; 20 min) and surface  (1.5% trypticase plates  were  inoculated of  was as follows. Spore  peptone,  incubated  1.0% yeast overnight  extract, 42°C.  onto  Germination  Agar  1.5% agar). Germination Single  colonies  were  into Cooked Meat medium, incubated 24 h, and a loopful  culture used to inoculate  without  at  plated  suspension was heat  c-galactosides). This  15 mL of o-Galactoside Broth (with or culture  was  incubated  overnight,  then  31 used as the turn,  inoculum for  after  6  h  125 mL of  incubation,  was  Broth, which in  o-Galactoside  the  inoculum  for  2,500  mL  of  o-Galactoside  Broth. Following germination of the Spores, no special  steps  taken  were  autoclaved  media  to was  ensure used  anaerobiosis,  throughout  and  except cultures  that  freshly  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  Filtration Gel  column with  filtration  of  collected  protein  ovalbumin, as  all  described  by  3. E N Z Y M E  Freifelder  (15  min)  phosphate  assay  Sigma  (St.  Que.),  activity  and  made  by  serum  Louis,  92  cm  equilibrated (6  mL)  were  absorbance  comparison  albumin  MO).  x  at  with  (BSA),  and  Calculations  were  (1976).  5  45°C.  (pH  read  of  6.5),  was  monitored  The  of  1.5  reaction  at  405 to  1 ug  mg  enzyme.  volumes  required  absorbance  activity at  amount  of  absorbance  4.  bovine  cm  6.7. F r a c t i o n s  were  (liver),  2.6  Dorval,  alpha-D-galactopyranoside  buffer  appropriate  amount  pH  a  ACTIVITY  p-nitrophenyl  addition  in  Inc.,  buffer,  estimations  by  o-Galactosidase time  2°C  o-galactosidase  catalase  supplied  at  (Pharmacia,  for  weight  standards  out  phosphate  assayed  Molecular  carried  S400  potassium  and  nm.  was  Sephacryl  0.05M  280  Chromatography  of  nm.  hydrolyse  M  unit 1  p-nitrophenol/mL  of  a  fixed  contained  0.67mM  (PNPG),  0.06M  sodium  3mM  reaction  0.1  means  mixture  BSA/mL,  The  One  by  cysteine,  was  sodium  stopped  carbonate  of  enzyme  umole  of  was  0.124.  and  was  by  the  and  the  defined  PNPG  per  an  as  min.  the The  ELECTROPHORESIS Sodium  (SDS-PAGE)  dodecyl was  carried  electrophoresis  unit  (1970).  bands  stain  Protein consisting  sulphate  of  (Atta were 27%  out Inc.,  in  polyacrylamide a  12  Japan)  located,  by  isopropanol,  cm by  x the  means 10%  gel 12  electrophoresis  cm  vertical  method of  acetic  a  of  Laemmli  Coomassie acid,  slab  and  blue 0.04%  33 Coomassie brilliant blue R-250 (BioRad, Richmond, CA) in water. Gels were stained overnight, then destained with acetic  acid  in water. Calculation  of  12% isopropanol  corresponding  and 7%  molecular  weights  was by the method described by Weber and Osborn, (1969).  5. ISOELECTRIC  FOCUSING  Analytical  horizontal  polyacrylamide  gel  (IEF-PAGE) was carried out in a Bio-Rad Model cell, according to  the  manufacturers  isoelectric  focusing  1415 electrophoresis  instructions. Gel  slabs were 45  mm x 125 mm and either 0.8 or  1.6 mm thick. Bands were  by  protein  means  of  a Coomassie  a-galactosidase paper  and  activity  flooded  assay .Following gel with  with  stain  with  the  incubation  sodium  a surface  blue  stain  in which the gel was solutions  stopped  in  (0.5 cm  probe;  a  PNPG  the  enzyme  by flooding the  solution. The pH gradient  pH electrode  by  placed on filter  described  the reaction was  carbonate  and/or  located  was  Corning  measured  Sci. Products,  Medfield, MA) and a Fisher Accumet pH meter, Model 620.  6. THERMAL  STABILITY  Duplicate incubated for  0.1  temperatures  incubation then  samples  of  purified  15 min at various temperatures  test- tubes. Test test  mL  tubes and  times were  immediately  were  pre-equilibrated  experiments equivalent  assayed  against  were  enzyme in  solution  12 mm x 100 mm  in water  staggered  to  to within 2 sec. The PNPG  at  were  baths  at the  ensure  that  enzyme was  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 velocities  time of  concentration  assays  enzyme  (1  min) were  substrate  reactions  8.0 ug protein/mL) at  used (pH  6 PNPG  to  estimate  initial  6.5; 40°C;  enzyme  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  and many complex  is frequently  difficult  to  induce  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 starch  in  and Rey (1979) reported  Duncan  recoveries  Strong  medium  with  in 6 out of 8 C. perfringens  12 of 21 strains gave higher spore Neither  that  replacement  raffinose  improved  of the spore  strains tested. In this study  counts  with raffinose  (Table 8).  carbohydrate, however, was preferred by all strains and one  strain failed to sporulate in either medium.  3. YIELDS OF o-GALACTOSIDASE When C. perfringens raffinose  M34 was grown in broth containing 0.5 %  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 and results utilizing  is known for its ability to grow rapidly at 45°C  presented here  a-galactosides  indicate  at  that  that  strain  M34 was capable of  temperature. The reason  42°C was  chosen for production of the enzyme was the possibility that genetic control of o-GALCp case  might  in C. perfringens  be plasmid mediated. Whether this  has not been  determined,  but  it  is the  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 al.,  1978).  In  light  of  the  possibility  of  of plasmids (Rood et  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  F1  400  F2  140  F4  40  F5  52  1580  F7  510  230  FA  6200  2400  M06  169  170  M20  10  160  M21 M22  a N.D. 330  M30  0.02  M31  2.7  M34 M40 M64 M74  800 0.18 a N.D. a N.D.  M75  250  M81  2600 a N.D.  M92 M24R a N.D.= none detected.  0.80  12.1 a N.D. 1450 6.7  24 1900 365 5.4 98 244 16 a N.D. 0.02 2.2 1800 82  38 enzyme, it was thought prudent to use a lower temperature to grow the organism.  4. PURIFICATION OF The  results  o-GALCP  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  activity  c-GALCp  at  a salt  concentration  of  0.19  M  (Figure 1).  The active  fractions were pooled, concentrated by ultrafiltration, and  applied  a  to  Sephacryl  S400  column. Active  fractions  were  pooled, concentrated, and reapplied to the same column. The activity  peak  from  this  column  purity acheived. SDS-PAGE  represents  the  highest  again pooled  degree  of  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  Sephadex G150 at room temperature resulted in loss of  a-GALCp  on  >60% of the  activity. A final attempt to improve purity by reapplying the sample to  DEAE  enzyme  cellulose activity.  resulted  Therefore  in  a  loss  further  of  greater  than  90% of  the  characterization  was  carried  out  with the Sephacryl S400(2) active fractions.  5. PH OPTIMUM Relative (Figure similiar.  3). The  rate  of  hydrolysis  pH optimum  of  of  PNPG  a-GALCp  was  greatest  in the  crude  at  pH 6.3  extract  was  39 Table 9. Effects of the purification steps on specific activity and yield of o-GALCp. Procedure  Activity  Specific  Yield  Activity (U/mL)  (U/mg  (%)  protein) French Press.  1.21  0.17  DEAE Cellulose  0.72  0.81  Sephacryl S400  0.18  Sephacryl S400 (2)  0.13  100 64 50  1.61  32  a-Galactosidase  Activity,  (U/mL)  OQ  c  ft 1  o  fl>  OQ P  •  h->  P  o  rt  n 3"  O  to H-  o  P  P  o.  3  c/> CD  rt **\ cw  P  o  rt  <  P  ^3 " X  o  rt  /  \  P. 1  CD  > > n  to o o c 3 o  >  m  o t—• »—•  o o  re  O tn  o ct>  0  3  3*  o.  <  c  t—'  o  to CD  3 O  (5 3  rt  H P  rt M-  o 3  P  cr t/>  o •i cr P  3 O  CD  Absorbance, <  (280  i  nm) o ON  Sodium  Chloride,  (M)  41  Figure and  2.  SDS-polyacrylamide  protein  s t a n d a r d s ; A,  S400 c h r o m a t o g r a p h y ;  B,  albumin ( p o l y m e r i z e d ) .  gel electrophoresis oC-GALCp peak  catalase;  C,  o f o(-GALCp  from S e p h a c r y l  bovine  serum  42  Figure  3.  pH  optimum  represents  the  o f ot-GALCp  average  at  30"C.  of duplicate  Each  point  determinations.  43  6. M O L E C U L A R The means  a  Although on  molecular appears  The  band  of  to  0.20  Burk  FOR  by ±  a  was  Vmax,  program  Vmax data  goodness  of  fit  (Figure  in  of  isolation  resulted  in  recoveries.  5). O n  identify  band  this  97,000 the  corresponded  basis,  the  native  on  a  pH  4.0  to  s t a i n , at  pH  pH  6.5  5.6±  gel  gradient  0.1. N o  other  the  or  minus  their  errors  were  Oestreicher  and  =  0.06  is  2.02  ±  included  6). L i n e a r  following  to  Pinto  (1983) to  uM/min.  allow  regression  parameter  standard  of  a  visual  the  values:  A  be;  Lineweaver estimate  Lineweaver  Km  Km  =  0.24  of  Burk mM;  uM/min.  9. A C T I V A T I O N  the  positively  prominent  (Figure  activity  of  mM; same  Loss  an  0.02  2.16  to  approximately  by  dimer.  plus  the  =  most  was  estimated  noted.  of  Vmax  S400,  as  PNPG  the  resulted  o-GALCp,  possible  the  focused  plot  plot  not  46,000  of  was  and  computed =  be  means  activity  Km  Sephacryl  was  of  of  POINT  by  8. A F F I N I T Y  it  weight  SDS-PAGE,  enzyme  located  on  weight  ISOELECTRIC  and  molecular  filtration  band  protein  7.  gel  4).  enzyme to  native  of  (Figure  WEIGHTS  OF  o-GALCP  enzyme  activity  procedure. loss  The  of  BY  at  microbial  Gel least  VARIOUS was  filtration 60%.  inhibitor  a  COMPOUNDS  recurrent  problem  room  temperature  at  Chromatography sodium  azide  at (0.02  2°C %)  throughout typically improved and  to  a  44  Figure  4.  Gel f i l t r a t i o n  m o l e c u l a r weight  chromatography  s t a n d a r d s on  o f oc-GALCp and  S e p h a c r y l S400 .  Figure  5.  weight  Elect.rophoretic standards  on  SDS  mobility  of  tf-GALCp  - polyacrylamide  and  gel.  molecular  -5.0  0  5.0  10.0  1/S Figure  6.  of  PNPG  min);  Linewe«ver hydrolysis  S«substrate  Burk at  pH  plot 6.5  o f o(-GALCp and  concentration  40*C: (mM).  kinetic  V-velocity  data (umoles/  47 lesser extent chlorhexidine gluconate (0.002 %) also decreased activity (Table 10). In assays at 45°C, 2-mercaptoethanol, NADH, cysteine, and BSA of  each increased the apparent activity of GALCp. The combination 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  2-mercaptoethanol  have  noted  and NADH  the  activation  on the a-galactosidase  effect  of  of E. coli K12  (Burstein and Kepes, 1971). The effect of BSA was examined because it  had  been  described  as  a-galactosidase of Bacillus 1980). It would  appear  improving  the  heat  stability  of  the  stearothermophilus (Pederson and Goodman,  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 serves  to  alters stabilize  the  active  site  the enzyme  former was the case  of  against  one would  the  enzyme,  or  whether  it  thermal  denaturation.  activity  to be increased at  expect  If the  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  100  96  122  mercaptoethanol 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 increased.  The  temperature purified  stability  (Figure 8). This apparent  enzyme  optimum  enzyme  enzyme  of  of  a-GALCp in  the  47°C  decreased crude  in a  extract  very  losses  likely during  accounts the  had  15 min assay  however, was 96% inactivated fact  markedly  by  an  (Figure  purity  apparent 7). The  15 min at 45°C  for at least  purification  as  some of the  procedure,  routine assay temperature during purification was 45°C.  as the  50  100  -  75 -  50 •  25  -  1 27  •—I  22  1 32  1 37  1 42  Temperature  gure  7.  (T)  Temperature and  in active  chromatography of  duplicate  optimums  i  i  52  57  (c)  of  fractions  ( • ) . Each  1, 47  cx-GALCp  of Sephacryl  point  determinations.  i n crude  represents  extract  S400 the  average  10  20  30  Temperature  ure  8.  various  Thernal  stability  temperatures  f o r 15  of  40  50  (*C)  oC-GALCp,  min,  then  incubated assayed  at  at 35°C.  52  D. CONCLUSIONS This study has reported the partial purification and characterization of from C. perfringens.  c-galactosidase weight Km  The enzyme has an apparent  of 96,000, an isoelectric point of pH 5.6, and a pH optimum of 6.3.  was 0.20 m M raffinose. Precise  useful  molecular  in elucidating the mechanism  enzyme  itself  could  animal  studies  of  perfringens  prove  information of  gas production  the flatulence  potential  of  legume  may be  . A l s o the  for in vitro and  components. The C.  be unsuitable  because of the pathogenic nature of C.  enzyme  in vivo  to be a valuable research tool  c-galactosidase, however, would  of human f o o d s  on this  for the treatment perfringens.  III. ISOLATION AND CHARACTERIZATION OF THE o-GALACTOSIDASE  OF  WASTE LAGER YEAST (SACCH AROMYCE S CARLS BE RGE NS/S).  A. INTRODUCTION treatment  a-Galactosidase removing  the  oligosaccharides  has  been  raffinose  legume products such as soybean milk 1976; Sugimoto treatment  can  and  suggested  as  a  stachyose  from  means legumes  of and  (Delente et al., 1974; Smiley et al.,  and van Buren, 1970; Cruz et al., 1981). A similiar enzyme be  used  to  remove  raffinose  from  sugar  beet  molasses,  thereby improving the crystallization yields of sucrose (Delente et al., 1974; Suzuki  et  al.,  inexpensive  1969). For  either  and toxiologically  application  unimpeachable  an  enzyme  source  is a important  which  is  prerequisite. In  this study it was found that waste lager yeast had relatively high levels of a-galactosidase probably  activity.  Waste  represents the cheapest  Furthermore, acceptable  brewers  source of  yeast another  is  brewers  yeast  is  in  plentiful  supply  and  source of this enzyme currently available. known  to  enzyme which  be  non-pathogenic  is approved for  and use  is  an  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  yeast (a-GALSc). .  53  a-galactosidase  from  waste  brewers  54 B. MATERIALS AND  1.  BREWERS  METHODS  YEAST  Waste  lager  yeast  was  Vancouver, B.C. The yeast was  donated  by  Molsons  Brewery,  identified by Molsons to be a strain  of Saccharomyces carlsbergensis .  2.  CRUDE  EXTRACT  Lager buffer,  pH  yeast  was  7.2, and  washed with  resuspended  in  3 volumes the  same  of  0.05M TRIS-HCI  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 Crude  of  1 to  extract  was  mixed with  1.6 volumes, immediately  '545'  (Fisher  then  resuspended  stirred (4°C,  4.  PRECIPITATION  Sci., Pittsburgh, PA) in  0.75  (6% by  volumes  of  weight  0.5M  of  Celite  crude extract),  TRIS-HCL  buffer  and  10 hours). Celite was removed by filtration.  Isopropanol extract  cm column of held at 2°C.  (-20°C) in a ratio  filtered thorough a pad of  SEPHADEX G100 GEL FILTRATION of  isopropanol  CHROMATOGRAPHY  (20 mL) was applied to a 2.5 cm x 70  Sephadex G100 (fine) (Pharmacia Inc., Dorval, Quebec) A  flow  rate  of  35 mL/h was  used. Fractions  were collected and a-galactosidase active fractions pooled.  (6 mL)  55 5.  ANION EXCHANGE  CHROMATOGRAPHY  A 2.5 cm x 22 cm column of Whattman DEAE cellulose DE-32 (Whatman  Chemical  Mcllvaine  buffer  fractions  from  applied  to  Seperation  (0.017M citric the  the  Sephadex  DEAE  Ltd.,  England)  acid and Na HP0 2  G100  cellulose  was  column  column,  equilibrated  with  to pH 5.6). Pooled  4  (100-300  washed  units)  with  300  were  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 Active  peaks  from  the  DEAE  CHROMATOGRAPHY cellulose  chromatography  were  concentrated by ultrafiltration at 4°C,  in an Amincon (Lexington, MA)  Model  PM-10  52 cell  fitted  with  a Diaflo  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 KH P0 -NaOH buffer (pH 6.7). 2  7. PROTEIN  MEASUREMENTS  Protein (1951),  as  4  determinations  modified  by  were  Peterson  by  the  (I977).  method  of  Crystalline  Lowry  et al.,  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  absorbance  of1  ug p-nitrophenol/mL  is  as  sufficient  defined  enzyme  at  was  to  405  nm  determined. The  0.124. One unit of  hydrolyze  1 umole  activity  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 carried out  gel  in a  electrophoresis  12 x12 cm  Inc., Japan) by the located acetic  by  means  acid,  and  vertical  method of of  a stain  0.04%  (SDS-PAGE).  slab electrophoresis  Laemmeli consisting  Coomassie  SDS-PAGE  was  unit  (Atto  (1970). Protein bands  were  of  27% isopropanol, 10%  brilliant  blue  R-250  (BioRad,  Richmond, CA). Gels were stained overnight, then destained with 12% isopropanol molecular  and weights  7%  acetic  was  as  acid.  described  Calculations by  Weber  of  corresponding  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,  glutaraldehyde by the method of Payne, (1973).  was  prepared  with  57  10.  ISOELECTRIC  FOCUSING  Analytical was  done  the  in  a  0.8  gradient  Corning  meter,  Model  enzyme  assay. the  ug  40°C  (20 with  second  carried were  fit  taken reaction  by  data  by  two  a  pH  probe  MA)  and  the  x  125  (0.5  a  mm  band  was  reaction  Regions  The  diameter  Accumet  pH  located  by  described was  and  used.  cm  Fisher  to  (BioRad,  were  solutions  the  according  ampholytes  carbonate.  yellow  in  the  stopped  by  of  o-GALSc  colour.  Vo  as  the  30  average  reaction  curve  product by  each  and  first  first  at  the  The  the  stoppers, curve  reaction  concentration  by  the  time  In of  reaction  bottles.  at the the was  Samples  analyzed  constructed. order  was  used  period.  derivative  serum  depletion the  first  fixed  were  that  zero.  glass  determining  concentration  instance,  over  time  through  concentrations  raffinose)  velocity from  substrate  substrate  the with  stoppered  syringes a  In  min  estimated  sterile  substrate  procedures.Enzyme  was  in  various  mixture.  PNPG;  aseptically with  at  different  reaction  taken  at  with  mm  gradient  galactosidase gel  1M  6.2  surface  velocities  with  accomplished the  o-  Biolyte  pH  Medfield,  45  cell,  (IEF-PAGE)  AFFINITY  enzyme out  to  a  focused  were  thick.  sodium  instance,  substrate  slabs  with  min Vo  Gel  focusing  electrophoresis  incubation,  protein/mL  assays  The  reaction  estimated  1415  3.7  with  indicated  Initial  0.1  pH  isoelectric  Following  gel  were  gel  mm  Products,  freshly  SUBSTRATE  were  a  620.  the  11.  with  Sci.  flooding  activity  1.6  measured  probe;  flooding  or  CA)  was  Model  instructions.  mm  Richmond,  was  BioRad  manufacturers  either  the  polyacrylamide  which  optimization  for This best 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  standard errors were computed with the program of  corresponding  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  (0.67mM) and  raffinose  (0.17M  acid,  citric  determined  as  on  activity  (20mM) as  0.2M  described  was  substrates,  Na P0 ,  pH  in  enzyme  2  4  the  examined  3.5  in  to  using  PNPG  Mcllvaine's  buffer  6.5).  assay.  Activity  Activities  was were  expressed as percentages of the activity at pH 4.5.  C. RESULTS AND DISCUSSION  1. YIELDS One units  of  gram (dry wt.) of a-GALSc  in the  washed, waste  clarified  wall fraction was not assayed for  2.  crude  lager yeast  extract  yielded 27  fraction. The  cell  o-GALSc.  PURIFICATION The  Table  11.  results The  of  the  purification  isopropanol  procedures  precipitation  step  are  summarized  provided  a  in  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  1.79  16.08  0.11  100  2-Propanol Prec  1.18  2.98  0.40  70  G100 Sephadex  1.11  0.18  6.17  56 45  DEAE Cellulose (total) DEAE (peak A)  0.10  (S400 peak A)  0.38  DEAE (peak B)  0.54  (S400 peak B)  0.53  DEAE (peak C)  0.46  (S400 peak C)  0.40  11 0.007  54.8 15  0.011  48.2 19  0.004  88.8  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 peak  a-GALSc  of  chromatographed  on  activity DEAE  from  Sephadex  S.  A-50.  when  carlsbergensis Consequently  one  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 to  have  a  molecular 95,000,  weight  118,000,  a-GALSc-C  weight  of  (Figure  10). BSA, catalase, ovalbumin, and  molecular  and  of  standards, a-GALSc-A  weight  well  documented  glycoproteins  of  (Andrews,  molecular  some  1965).  a  weight  y-globulin  r-Globulin  standards.  anomalously, a characteristic  a  a-GALSc-B  a-GALSc  (Lazo, 1977), and the possibility  molecular of  were  behaved  glycoproteins  appeared  65,000 used as  somewhat  which has been  enzymes  are  also  that this affected the  gel filtration charactistics can not be excluded.  3.  SODIUM  DODECYL  SULFATE  POLYACRYLAMIDE  GEL  ELECTROPHORESIS a-GALSc SDS-PAGE  peaks  profiles  were  (Figure  also  11). A  distinctive diffuse,  band corresponding to molecular weights  in  poorly  terms  of  resolved  of approximately  their protein  65,000 to  62  ure  10.  and  Gel f i l t r a t i o n  protein  standards  o f <x-GALSc-A, on  Sephacryl  ac-GALSc-B, S400.  a(-GALSc-C  63  gure  11.  SDS-polyacrylamide  fractions ol-GALSc  and b o v i n e  peak  from  DEAE c e l l u l o s e  Sephadex  column;  column; E, ct-GALSc-C serum  albumin  serum  gel electrophoresis a l b u m i n : A, c r u d e  extract;  G100 column; C, <*-GALSc-A  D, cX-GALSc-B from  from  o f a(-GALSc  DEAE c e l l u l o s e  (polymerized).  DEAE  B, from  cellulose  column;  F, b o v i n e  64 130,000  can  be  seen  to  purification process. DEAE to  correspond  to  be  progressively . intensified  cellulose  large,  by  the  peaks A, B and C can be seen  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 peaks  a-GALSc the  active  bands  A, B and C were  detected  by means  of  focused  by  IEF-PAGE and  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 a  carlsbergensis,  large  extracellular  one form  of  glycoprotein  a-GALSc  with  a  in 5.  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  Saccharomyces is suggested  extensively invertase  as a model  (Lazo  studied et  for the study  exocellular  al., 1977). of  enzyme  Invertase  glycoprotein  has  of been  synthesis and  secretion by yeasts (Moreno et al., 1975; Gascon et al., 1968). In the case of invertase, multiple external enzyme  molecular forms have been detected. The  is apparently homogeneous, with a molecular weight  65 of approximately have  been  suggested addition  270,000, while at least three smaller  reported that  of  the  (Moreno internal  carbohydrate  et  al.,  forms units  authors have suggested that  1975).  of  to  Moreno  invertase a  et  al.  represent  common  the multiple  internal forms  forms  (1975)  sequential  polypeptide. may  be  Other  degradation  products and that only two true forms exist; an internal protein and a external glycoprotein (Lazo et al., 1977). The relative proportions of been  shown  to  was  cultured.  depend The  upon  larger,  internal and external invertase have  the  conditions  external  form  under  which  the  predominates  yeast  when  the  invertase system is induced. (Sutton and Lampen, 1962). The system  information  may  be  presented  comparable  to  here the  suggests  invertase  that  the  system. The  a-GALSc 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  determine, first  of  external  whether  enzymes.  an external  Further enzyme  study  is  required  to  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  examined  (Figure  raffinose same  was  pH  of  12).  optimum using  activity  of  at  pH 4.5. Lazo  for  the  PNPG  as  o-GALSc-C  activity  a-Galactosidase  maximum  carlsbergensis,  the  et  a  towards  pH  was  PNPG  and  al. (1977) reported  they  a-GAL  to  substrate.  isolated Similar  the  from  results  S. 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  PNPG at pHs above the optimum. Since activity towards sugar  raffinose  processing  higher  a-GALSc  where  more  acidic  towards  showed substantial  at pH 5 to 5.5, it might  industry  than  find use in the  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 and pH 4.5, for  both PNPG  (Vo)  estimated  was  first  and raffinose at  different  (Table  at 40°C  12). Initial velocity  substrate  concentrations  by  fixed time assays. This linearity  in  assumption validate the  method the that  of  reaction is  of  curve  frequently  experimentally  assumption  determining up  Vo to  requires the  difficult  to  point  an of  justify  assumption the  assay, an  theoretically  (Allison and Purich, 1979). In practical  linearity  may  decrease  and Vmax calculations. In an attempt  the  precision  to circumvent  of  of  or  terms, the Km  the problem, an  alternate approach was used to calculate Vo in which Vo values were  67  ure  12.  PNPG point  The ( •  )  effect and  of  pH  raffinose  represents  the  on  activity  ( O  average  ) of  as  of  a-GALSc-C  substrates.  duplicate  with  Each  determinations.  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) Vmax (uM/min) RSE (uM/min)  Raffinose  2.58  2.54 ±0.32 21.4 ± 1.4  23.8  3.10 x 10-  1  ±0.11 ±0.5  1.13 x 10-'  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 1 0  1  69 derived  from  (Figure  experimentally  13). The  equations  determined which  best  enzyme  reaction  the  experimental  fit  curves data,  together with the calculated Vo values are presented in Table 13. Km  and  determinations  Vmax by  values  the  were  method  of  computed Oestreicher  for  each  set  and Pinto  of  Vo  (1983). This  method was preferred to the usual graphic analysis for two reasons. The  Lineweaver  Burke  deceptive  because  plot  of  are  of  equal  varience  varies  Askelof  et  Plot, although the inherent  weight,  when  directly  with  1976;  Nimmo  al.,  emminently  convenient, can be  assumption that  all points  in  been  fact  reaction and  it  has  velocity  Mabood,  (Storer  in the  shown et  1979). The  that  al., 1975; Oestreicher  Pinto (1983) procedure employed weighting factors  ( inverse velocity  squared)  Lineweaver  to  procedure  compensate  provided  no  for  this.  estimate  Secondly, of  the  the  reliability  of  the  Burke 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  standard errors were decreased by the curve fitting method.  residual  70  Time ( h r )  Figure  13.  4.5,  Hydrolysis and  5  of  different  PNPG  by  i n i t i a l  d-GALSc-C PNPG  at  40"C,  pH  concentrations.  71  Table 13. Results of curve fitting to o-GALSC kinetic data at various initial substrate concentrations. Substrate  Initial  R e g r e s s i o n Equation  R  2  Derived  (umoles/min)  Cone.  (mM) PNPG  Raffinose  Vo  0.24  Iny -  0.48  y-(M  1.00  y-l  2.00  y-tt7  4.00  y-l  4.00  Iny = -0.20x  6.67  -0.84x -  1.18  0.9997  2.01  + 1.34  1.0000  3.77  0.9999  6.50  0.9997  10.56  0.9999  14.46  + 1.39  0.9932  8.2  Iny = -0.18X  + 1.90  0.9932  12.0  20.0  Iny = -0.11x  + 3.00  0.9967  22.1  40.0  y-l  0.9993  29.5  = 0.42x = 0.65x = 0.23x = 0.09x  + 1.00 + 0.62 + 0.25  = 0.0018X  + 0.025  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  yeast  make  demonstrated  it  achieved  ideal  that  disruption-solvent be  an  cost, availability commercial can  a-GALSc  and proven safety of  enzyme be  source.  recovered  This  by  a  brewers  study  has  rapid  cell  precipitation procedure. Further purification, if desired can  by  gel  chromotography. This any  low  a-galactosidase  filtration  enzyme  and/or  source  requirement  in  DEAE  could the  cellulose  therefore  food  be  ion  exchange  recommended  industry, particularity  intended substrate pH is in the range of optimum a-GALSc  activity.  if  for the  IV. REFERENCES  Adya,  S.  and  Elbein,  A.D.  1977.  Glycoprotein  Aspergil lus niger; purification  and  enzymes  properties  of  secreted  by J.  a-galactosidase.  Bacteriol. 129: 850. Akiba,  T.  and  Horikoshi,  K.  1976.  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