CA1131142A - Glucoamylase from stachybotrys subsimplex - Google Patents
Glucoamylase from stachybotrys subsimplexInfo
- Publication number
- CA1131142A CA1131142A CA332,250A CA332250A CA1131142A CA 1131142 A CA1131142 A CA 1131142A CA 332250 A CA332250 A CA 332250A CA 1131142 A CA1131142 A CA 1131142A
- Authority
- CA
- Canada
- Prior art keywords
- glucoamylase
- enzyme
- dextrose
- enzyme preparation
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 102100022624 Glucoamylase Human genes 0.000 title claims abstract description 58
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 title claims abstract description 43
- 241001598066 Brevistachys subsimplex Species 0.000 title claims description 9
- 238000000034 method Methods 0.000 claims abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims description 81
- 102000004190 Enzymes Human genes 0.000 claims description 81
- 229920002472 Starch Polymers 0.000 claims description 30
- 235000019698 starch Nutrition 0.000 claims description 30
- 239000008107 starch Substances 0.000 claims description 29
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 24
- 239000008121 dextrose Substances 0.000 claims description 24
- 239000002609 medium Substances 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 230000000694 effects Effects 0.000 claims description 13
- 239000007787 solid Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 8
- 229920005654 Sephadex Polymers 0.000 claims description 4
- 239000012507 Sephadex™ Substances 0.000 claims description 4
- 229920002774 Maltodextrin Polymers 0.000 claims description 3
- 239000005913 Maltodextrin Substances 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 claims description 3
- 230000004151 fermentation Effects 0.000 claims description 3
- 229940035034 maltodextrin Drugs 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 2
- 239000006188 syrup Substances 0.000 claims description 2
- 235000020357 syrup Nutrition 0.000 claims description 2
- 241001279361 Stachybotrys Species 0.000 claims 1
- 238000004440 column chromatography Methods 0.000 claims 1
- 238000012258 culturing Methods 0.000 claims 1
- 235000015097 nutrients Nutrition 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 229940088598 enzyme Drugs 0.000 description 80
- 108050008938 Glucoamylases Proteins 0.000 description 21
- 239000000243 solution Substances 0.000 description 20
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 16
- 244000005700 microbiome Species 0.000 description 14
- 239000000872 buffer Substances 0.000 description 12
- 230000007935 neutral effect Effects 0.000 description 10
- 239000007853 buffer solution Substances 0.000 description 9
- 239000000758 substrate Substances 0.000 description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 8
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 229960004592 isopropanol Drugs 0.000 description 8
- 241000233866 Fungi Species 0.000 description 7
- 241000228212 Aspergillus Species 0.000 description 6
- 241000235527 Rhizopus Species 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 5
- 240000007594 Oryza sativa Species 0.000 description 5
- 235000007164 Oryza sativa Nutrition 0.000 description 5
- 240000008042 Zea mays Species 0.000 description 5
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 235000005822 corn Nutrition 0.000 description 5
- 230000002779 inactivation Effects 0.000 description 5
- 235000009566 rice Nutrition 0.000 description 5
- 239000011345 viscous material Substances 0.000 description 5
- 241000228245 Aspergillus niger Species 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 101710108470 Hyalin Proteins 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000012225 czapek media Substances 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 210000004276 hyalin Anatomy 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 235000012343 cottonseed oil Nutrition 0.000 description 3
- 239000002385 cottonseed oil Substances 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 229920001353 Dextrin Polymers 0.000 description 2
- 239000004375 Dextrin Substances 0.000 description 2
- 229920002245 Dextrose equivalent Polymers 0.000 description 2
- 229920002527 Glycogen Polymers 0.000 description 2
- 241001330975 Magnaporthe oryzae Species 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 241000729876 Niveus Species 0.000 description 2
- 239000004373 Pullulan Substances 0.000 description 2
- 229920001218 Pullulan Polymers 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 108090000637 alpha-Amylases Proteins 0.000 description 2
- 102000004139 alpha-Amylases Human genes 0.000 description 2
- 229940024171 alpha-amylase Drugs 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000001058 brown pigment Substances 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 235000011148 calcium chloride Nutrition 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000010908 decantation Methods 0.000 description 2
- 235000019425 dextrin Nutrition 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000005755 formation reaction Methods 0.000 description 2
- 229940096919 glycogen Drugs 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000002932 luster Substances 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 235000019423 pullulan Nutrition 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- DBTMGCOVALSLOR-UHFFFAOYSA-N 32-alpha-galactosyl-3-alpha-galactosyl-galactose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(OC2C(C(CO)OC(O)C2O)O)OC(CO)C1O DBTMGCOVALSLOR-UHFFFAOYSA-N 0.000 description 1
- OQVYMXCRDHDTTH-UHFFFAOYSA-N 4-(diethoxyphosphorylmethyl)-2-[4-(diethoxyphosphorylmethyl)pyridin-2-yl]pyridine Chemical class CCOP(=O)(OCC)CC1=CC=NC(C=2N=CC=C(CP(=O)(OCC)OCC)C=2)=C1 OQVYMXCRDHDTTH-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 229920000945 Amylopectin Polymers 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241001619326 Cephalosporium Species 0.000 description 1
- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- ZCLAHGAZPPEVDX-UHFFFAOYSA-N D-panose Natural products OC1C(O)C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC1COC1C(O)C(O)C(O)C(CO)O1 ZCLAHGAZPPEVDX-UHFFFAOYSA-N 0.000 description 1
- 241000178951 Endomyces Species 0.000 description 1
- 241000502261 Endomyces sp. Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- AYRXSINWFIIFAE-SCLMCMATSA-N Isomaltose Natural products OC[C@H]1O[C@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)[C@@H](O)[C@@H](O)[C@@H]1O AYRXSINWFIIFAE-SCLMCMATSA-N 0.000 description 1
- 241001590997 Moolgarda engeli Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000952054 Rhizopus sp. Species 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 244000157378 Rubus niveus Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000872198 Serjania polyphylla Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000223261 Trichoderma viride Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- OCIBBXPLUVYKCH-QXVNYKTNSA-N alpha-maltohexaose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O[C@@H]2[C@H](O[C@H](O[C@@H]3[C@H](O[C@H](O[C@@H]4[C@H](O[C@H](O[C@@H]5[C@H](O[C@H](O)[C@H](O)[C@H]5O)CO)[C@H](O)[C@H]4O)CO)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O OCIBBXPLUVYKCH-QXVNYKTNSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- -1 carbohydrate compounds Chemical class 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000008504 concentrate Nutrition 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 235000021186 dishes Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229960002089 ferrous chloride Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910001959 inorganic nitrate Inorganic materials 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical class Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- DLRVVLDZNNYCBX-RTPHMHGBSA-N isomaltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-RTPHMHGBSA-N 0.000 description 1
- DJMVHSOAUQHPSN-UHFFFAOYSA-N malto-hexaose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(OC4C(C(O)C(O)C(CO)O4)O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 DJMVHSOAUQHPSN-UHFFFAOYSA-N 0.000 description 1
- FYGDTMLNYKFZSV-UHFFFAOYSA-N mannotriose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(O)C(O)C2O)CO)C(O)C1O FYGDTMLNYKFZSV-UHFFFAOYSA-N 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical class Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 229910052751 metal Chemical class 0.000 description 1
- 239000002184 metal Chemical class 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- ZCLAHGAZPPEVDX-MQHGYYCBSA-N panose Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@@H]1CO[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 ZCLAHGAZPPEVDX-MQHGYYCBSA-N 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- FYGDTMLNYKFZSV-BYLHFPJWSA-N β-1,4-galactotrioside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-BYLHFPJWSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/20—Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Process for the production of a glucoamylase having a pH optimum at about 6.0 to 6.5 by a strain of Sachybotrys and the glucoamylase produced thereby.
Process for the production of a glucoamylase having a pH optimum at about 6.0 to 6.5 by a strain of Sachybotrys and the glucoamylase produced thereby.
Description
~1311'.~Z
BACKGROUND OF THE INVENTION
At present, when producing dextrose indus-trially from starch, the principal glucoamylases employed for the saccharification process are those produced by micro-organism belonging to the genera Rhizopus and Aspergillus.
The conditions under which these glucoamylases are employed are pH 5.0 and 55C for the enzyme of the Rhizopus micro-organism, and pH 4.5 and 60C for the Aspergillus micro-organism's enzyme. In addition, maximum dextrose content of the hydrolyzate is about 96% (dry solids basis) when these glucoamylases react with enzyme liquefied starch at a 30% concentration. One reason that the dextrose yield does not reach 100% is that isomaltose accumulates due to a reverse reaction by these glucoamylases. However, there was recently published a report (U.S. Patent 3,897,305) that the reverse reaction of glucoamylses is extremely small in the vicinity of neutrality and that the dextrose yield can thus be elevated to about 98% by carrying out the reaction at about a neutral pH with the joint use of pullalanase. The pullalanase acts to debranch the starch and increases the rate of glucoamylase action under these nearly neutral conditions. As far as neutral glucoamylases are concerned, only one has ~een reported to date, that being the glucoamylase produced by the rice blast-causing fungus (Piricularia oryzae; Kazuo Matsuda, et al:
Amylase Symposium, Vol. 9, 1974), but t~ls glucoamylase possesses low thermostability and so cannot be employed under industrial conditions.
SUMMARY OF THE INVENTION
It is therefore a primary object of the present invention to provide a glucoamYlase that is active at nearly neutral pH.
It is another object of the invention to provide a glucoamylase that possesses enough thermostability so that it can be employed under industrial reaction conditions.
It is yet another object of the invention to provide a glucoamylase that reacts with a starch hydrolyzate to give high yields of dextrose.
A microbial strain has been discovered which produces a new glucoamylase having optimum activity at a pH of 6.o to 6.5 and good thermostability. The new glucoamylase is capable of converting a 30% by weight solution of a 10 D.E. (dextrose equivalent) liquefied starch to a product containing at least about 96% dextrose when reacted with the starch hydrolyzate at pH 6.0 to 6.5 at 55C. This invention includes the method for the production o~ this glucoamylase wherein the microorganism 11;31142 of the genus Stach,ybotrys, which produces the glucoamy,ase, is cultured in a medium and the enzyme is recovered from ' the culture broth.
.
BRIEF DESCRIPTION OF THE DRA~INGS
Figure 1 shows the relationship between the pH
and the enzyme activity in the cases of the enzyme of the present invention and the conventional glucoamylases produced by _. niveus and A. niger microorganisms.
Figure 2 shows the relationship between the temperature and the enzyme activity in the cases of the present enzyme and the glucoamylase from P. ~
Figure 3 presents the inactivation curves for the enzyme of this invention when it is treated at various 'pH levels.
Figure 4 provides a comparison of the present enzyme and the conventional glucoamylases produced by the R. ni'veus, A. niger and P. oryzae microorganisms in terms of their relative thermostabilities.
DETAILED DESCRIPTION~ OF THE INVENTION
The properties of the novel neutral gluco-amylase of the present invention are presented in detail~
and their properties are contrasted with those of the previously-known glucoamylases.
The term "D.E." is an abbreviation for "dextrose equivalent", and these terms are used interchangeably to refer to the reducing sugar content of a material calculated as dextrose and expressed as percent of total solids.
The term "starch hydrolyzate" is used in a general way to refer to a syrup or dry product that is made by the partial hydrolysis of starch. Such a product may be made by acid or enzymic hydrolysis.
The term "liquefied starch" is used to refer to a low D.E. (D.E. from about 2 to about 20) starch hydrolyzate.
1. Activity and Substrate Specificity The present enzyme is able to hydrolyze such carbohydrate compounds as starch, soluble starch, amylose, amylopectin and glycogen, and to produce dextrose from them. The yield of dextrose from each of these substrates is 100% when the substrate concentration is 1%. The muta-rotation of the produced dextrose is positive. This enzyme is thus a glucoamylase. The reaction velocity of this enzyme was compared to the rates shown by the glucoamylases produced by microorganisms belonging to Rhizopus and Aspergillus in relation to various substrates. The results .
are presented in Table I. As can be seen from this table, the activity of the present enzyme is notably higher than the activities of the other two glucoamylases especially in relation to the hydrolysis of pullulan.
Z
TABLE I
SUBSTRATE SPECIFICITY
; Reaction Ratea) AspergillusRhizopus niger niveus Present Gluco- Gluco-SubstrateEnzymeamylaseb)amylaseC) Dextrin (D.E. 10) 100 100 100 .
Amylpectin 104 113 91 Soluble Starch 122 95 112 Pullulan 9 2 2 Glycogen 102 100 91 Maltotriose 6 12 8 '! Maltohexaose~1 100 146 Panose 44 47 48 Maltose 14 26 19 ' . , , a) The enzymatic activitie$ of each glucoamylase were determined with the sub.strates present at a 1% con-centration; each enzyme t S acti.vity in relation to dextrin was assigned the value 100, and the activities on the other substrates are presented as relative - values.
b) Available from Enzyme Development Corporation,
BACKGROUND OF THE INVENTION
At present, when producing dextrose indus-trially from starch, the principal glucoamylases employed for the saccharification process are those produced by micro-organism belonging to the genera Rhizopus and Aspergillus.
The conditions under which these glucoamylases are employed are pH 5.0 and 55C for the enzyme of the Rhizopus micro-organism, and pH 4.5 and 60C for the Aspergillus micro-organism's enzyme. In addition, maximum dextrose content of the hydrolyzate is about 96% (dry solids basis) when these glucoamylases react with enzyme liquefied starch at a 30% concentration. One reason that the dextrose yield does not reach 100% is that isomaltose accumulates due to a reverse reaction by these glucoamylases. However, there was recently published a report (U.S. Patent 3,897,305) that the reverse reaction of glucoamylses is extremely small in the vicinity of neutrality and that the dextrose yield can thus be elevated to about 98% by carrying out the reaction at about a neutral pH with the joint use of pullalanase. The pullalanase acts to debranch the starch and increases the rate of glucoamylase action under these nearly neutral conditions. As far as neutral glucoamylases are concerned, only one has ~een reported to date, that being the glucoamylase produced by the rice blast-causing fungus (Piricularia oryzae; Kazuo Matsuda, et al:
Amylase Symposium, Vol. 9, 1974), but t~ls glucoamylase possesses low thermostability and so cannot be employed under industrial conditions.
SUMMARY OF THE INVENTION
It is therefore a primary object of the present invention to provide a glucoamYlase that is active at nearly neutral pH.
It is another object of the invention to provide a glucoamylase that possesses enough thermostability so that it can be employed under industrial reaction conditions.
It is yet another object of the invention to provide a glucoamylase that reacts with a starch hydrolyzate to give high yields of dextrose.
A microbial strain has been discovered which produces a new glucoamylase having optimum activity at a pH of 6.o to 6.5 and good thermostability. The new glucoamylase is capable of converting a 30% by weight solution of a 10 D.E. (dextrose equivalent) liquefied starch to a product containing at least about 96% dextrose when reacted with the starch hydrolyzate at pH 6.0 to 6.5 at 55C. This invention includes the method for the production o~ this glucoamylase wherein the microorganism 11;31142 of the genus Stach,ybotrys, which produces the glucoamy,ase, is cultured in a medium and the enzyme is recovered from ' the culture broth.
.
BRIEF DESCRIPTION OF THE DRA~INGS
Figure 1 shows the relationship between the pH
and the enzyme activity in the cases of the enzyme of the present invention and the conventional glucoamylases produced by _. niveus and A. niger microorganisms.
Figure 2 shows the relationship between the temperature and the enzyme activity in the cases of the present enzyme and the glucoamylase from P. ~
Figure 3 presents the inactivation curves for the enzyme of this invention when it is treated at various 'pH levels.
Figure 4 provides a comparison of the present enzyme and the conventional glucoamylases produced by the R. ni'veus, A. niger and P. oryzae microorganisms in terms of their relative thermostabilities.
DETAILED DESCRIPTION~ OF THE INVENTION
The properties of the novel neutral gluco-amylase of the present invention are presented in detail~
and their properties are contrasted with those of the previously-known glucoamylases.
The term "D.E." is an abbreviation for "dextrose equivalent", and these terms are used interchangeably to refer to the reducing sugar content of a material calculated as dextrose and expressed as percent of total solids.
The term "starch hydrolyzate" is used in a general way to refer to a syrup or dry product that is made by the partial hydrolysis of starch. Such a product may be made by acid or enzymic hydrolysis.
The term "liquefied starch" is used to refer to a low D.E. (D.E. from about 2 to about 20) starch hydrolyzate.
1. Activity and Substrate Specificity The present enzyme is able to hydrolyze such carbohydrate compounds as starch, soluble starch, amylose, amylopectin and glycogen, and to produce dextrose from them. The yield of dextrose from each of these substrates is 100% when the substrate concentration is 1%. The muta-rotation of the produced dextrose is positive. This enzyme is thus a glucoamylase. The reaction velocity of this enzyme was compared to the rates shown by the glucoamylases produced by microorganisms belonging to Rhizopus and Aspergillus in relation to various substrates. The results .
are presented in Table I. As can be seen from this table, the activity of the present enzyme is notably higher than the activities of the other two glucoamylases especially in relation to the hydrolysis of pullulan.
Z
TABLE I
SUBSTRATE SPECIFICITY
; Reaction Ratea) AspergillusRhizopus niger niveus Present Gluco- Gluco-SubstrateEnzymeamylaseb)amylaseC) Dextrin (D.E. 10) 100 100 100 .
Amylpectin 104 113 91 Soluble Starch 122 95 112 Pullulan 9 2 2 Glycogen 102 100 91 Maltotriose 6 12 8 '! Maltohexaose~1 100 146 Panose 44 47 48 Maltose 14 26 19 ' . , , a) The enzymatic activitie$ of each glucoamylase were determined with the sub.strates present at a 1% con-centration; each enzyme t S acti.vity in relation to dextrin was assigned the value 100, and the activities on the other substrates are presented as relative - values.
b) Available from Enzyme Development Corporation,
- 2 Penn Plaza, New York, N.Y.
c) 5umyzyme available from Sumitomo Shoji Kaisha, Ltd., l, Kanda Mitoshiro-Cho, Chiyoda-ku, Tokyo, Japan.
2. Optimum pH and Stable pH Range The relationship between the enzymatic activity (relative value) of the present enzyme and the reaction pH were investigated and then compared with the corre-sponding relationships for the conventionally-known glucoamylases produced by the Rhizopus and Aspergillus microorganisms. The results are presented in Figure 1.
114;~
As shown in the figure, the optlmum pH of this enz~me at 60C is 6.0 to 6.5, considerably higher than the pH optima of the other enzymes. In addition, this enzyme shows its best stability in the vicinity of pH 6.o, but no inacti-vation of this glucoamylase is seen even when it is left sitting for 24 hours at room temperature, over a pH range of 4 to 11.
c) 5umyzyme available from Sumitomo Shoji Kaisha, Ltd., l, Kanda Mitoshiro-Cho, Chiyoda-ku, Tokyo, Japan.
2. Optimum pH and Stable pH Range The relationship between the enzymatic activity (relative value) of the present enzyme and the reaction pH were investigated and then compared with the corre-sponding relationships for the conventionally-known glucoamylases produced by the Rhizopus and Aspergillus microorganisms. The results are presented in Figure 1.
114;~
As shown in the figure, the optlmum pH of this enz~me at 60C is 6.0 to 6.5, considerably higher than the pH optima of the other enzymes. In addition, this enzyme shows its best stability in the vicinity of pH 6.o, but no inacti-vation of this glucoamylase is seen even when it is left sitting for 24 hours at room temperature, over a pH range of 4 to 11.
3. Determination_of Potency A 0.5-ml aliquot of a suitably diluted enzyme solution was added to 0.5 ml of a 2% solution of a spray-dried maltodextrin (D.E. about 10) in 0.1 M acetate buffer solution (pH 6.o) and this was incubat~ed at 60C for precisely 10 minutes. The enzyme reaction was then stopped by heating the mixture for 5 minutes in a boiling water ,bath. The amount of dextrose produced was determined by the glucose oxidase method. The amount of enzyme producing 1 micromole of dextrose per minute was defined as 1 unit.
4. Optimum Reaction Temperature Range The effect of temperature on the relative enzymatic activity of the present enzyme at pH 6,o was compared with, the relative activity for the knowr. glucoamylase from the rice blast fungus, Piricularia oryzae. This comparison is shown in Figure 2. It is evident that the optimum temperature for the reaction of the present enzyme under these conditions is ~5C, about 10C higher than that of the enzyme from Piricularia oryzae.
ll~ Z
ll~ Z
5. Inactivation Due to pH and Tem~erature Conditions .
- Figure 3 presents inactivation curves of the relative enzymatic activity of the present enzyme when it was treated for 60 minutes at 60C over a pH range of 3 to 8. As is clear from the figure, this enzyme is most stable at pH 6, and it is completely inactivated by this treatment for 30 minutes at pH 3 and for 1 hour at pH 4. In addition, Figure 4 shows a comp~rison of the thermostability of the present enzyme and the glucoamylases from the Rhizopus, Aspergillus and Plricularia microorganlsms. Namely, Figure 4 presents the inactivation curves obtained for these enzymes when they were treated at 60C while being held at their respective optimum pH's for stability. It can be seen that the thermostability of the present enzyme is inferior to that of the glucoamylase of Aspergillus - origin, but is superior to the thermostability shown by the glucoamylases from the Rhizopus and the Piricularia micro-organisms.
- Figure 3 presents inactivation curves of the relative enzymatic activity of the present enzyme when it was treated for 60 minutes at 60C over a pH range of 3 to 8. As is clear from the figure, this enzyme is most stable at pH 6, and it is completely inactivated by this treatment for 30 minutes at pH 3 and for 1 hour at pH 4. In addition, Figure 4 shows a comp~rison of the thermostability of the present enzyme and the glucoamylases from the Rhizopus, Aspergillus and Plricularia microorganlsms. Namely, Figure 4 presents the inactivation curves obtained for these enzymes when they were treated at 60C while being held at their respective optimum pH's for stability. It can be seen that the thermostability of the present enzyme is inferior to that of the glucoamylase of Aspergillus - origin, but is superior to the thermostability shown by the glucoamylases from the Rhizopus and the Piricularia micro-organisms.
6. Inhibition, Activation and Stabilization This enzyme does not require any special acti-vating or stabilizing agents. However, the same as in the case of most of the other glucoamylases, this enzyme is inhibited by mercuric chloride, potassium manganate, ferrous chloride, other metal salts and ~ris.
7. Purification Procedure The present enzyme can be purified by means of a combination of any of the ordïnary purification methods ~13,1~
such as ammonium sulfat~ fractianation, organic solvent fractionation, starch adsorption, and various chromatog~
raphies. An lllustrative example ~f such a purification procedure is presented next.
The cells and other insoluble material are eliminated from the cultured material and then the culture fluid is frozen overnight at -20C. This is then melted at room temperature and the insoluble matter is removed by centrifugation. Next, two volumes of cold isopropanol is added to this and it is left standing for one night at 4C. The enzyme precipltates and the supernatant is removed by decantation, The precipitate is then dissolved in a 0.05 M tris-HCl ~uffer solution (pH 7.5) containing 1 mM EDTA, and the dissolved material is next dialyzed for one night at 4C against the same buffer. DEAE-cellulose which has been equilibrated with the same buffer solution is next added to this dialyzed enzyme solution so that the enzyme is adsorbed thereto.
After washing this DEAE-cellulose with the same buffer, enzyme is eluted from the resin with a preparation of the same buffer containing 0.3 M NaCl. The enzyme is then precipitated by the addition of two volumes of cold iso-propanol to the eluate, and this precipitated material is recovered by centrifugation. The precipitate is dissolved in the 0.05 M tris-HCl buffer solution (pH 7.5) containing 1 mM EDTA, followed by overnight dialysis Z
against the same buffer. The dialyzed enzyme solution is ; next applied to a DEAE-cellulose column which has been equilibrated with the same 0.05 M tris-HCl buffer (pH
7.5) containing 1 mM EDTA. The enzyme is then eluted from this column by passing through it a linear concen-tration gradient of the same buffer containing MaCl up to 0.5M. The eluted fractions which contain the enzyme are pooled and the enzyme is concentrated by means of the isopropanol precipitation technique. This concentrated enzyme is then applied to a column of Sephadex* G-150 which has been equilibrated with a 0.05 M tris-HCl buffer (pH 7.0) containing 1 mM EDTA, and elution is carried out with the same buffer solution. When this procedure was followed, the purified enzyme which was obtained showed a single band in disc electrophoresis.
such as ammonium sulfat~ fractianation, organic solvent fractionation, starch adsorption, and various chromatog~
raphies. An lllustrative example ~f such a purification procedure is presented next.
The cells and other insoluble material are eliminated from the cultured material and then the culture fluid is frozen overnight at -20C. This is then melted at room temperature and the insoluble matter is removed by centrifugation. Next, two volumes of cold isopropanol is added to this and it is left standing for one night at 4C. The enzyme precipltates and the supernatant is removed by decantation, The precipitate is then dissolved in a 0.05 M tris-HCl ~uffer solution (pH 7.5) containing 1 mM EDTA, and the dissolved material is next dialyzed for one night at 4C against the same buffer. DEAE-cellulose which has been equilibrated with the same buffer solution is next added to this dialyzed enzyme solution so that the enzyme is adsorbed thereto.
After washing this DEAE-cellulose with the same buffer, enzyme is eluted from the resin with a preparation of the same buffer containing 0.3 M NaCl. The enzyme is then precipitated by the addition of two volumes of cold iso-propanol to the eluate, and this precipitated material is recovered by centrifugation. The precipitate is dissolved in the 0.05 M tris-HCl buffer solution (pH 7.5) containing 1 mM EDTA, followed by overnight dialysis Z
against the same buffer. The dialyzed enzyme solution is ; next applied to a DEAE-cellulose column which has been equilibrated with the same 0.05 M tris-HCl buffer (pH
7.5) containing 1 mM EDTA. The enzyme is then eluted from this column by passing through it a linear concen-tration gradient of the same buffer containing MaCl up to 0.5M. The eluted fractions which contain the enzyme are pooled and the enzyme is concentrated by means of the isopropanol precipitation technique. This concentrated enzyme is then applied to a column of Sephadex* G-150 which has been equilibrated with a 0.05 M tris-HCl buffer (pH 7.0) containing 1 mM EDTA, and elution is carried out with the same buffer solution. When this procedure was followed, the purified enzyme which was obtained showed a single band in disc electrophoresis.
8. Molecular Weight The molecular weight of the present enzyme was investigated using a Sephadex G-150 column in accordance with the procedure of Andrews, P., Biochem J. 96, 595 (1965). The results indicated that this enzyme's molecular weight is about 50,000.
Next, the points of difference between the present enzyme and the conventionally-known glucoamylases will be presented, and an explanation will be made of the reasons that this enzyme is to be considered a new enzyme having its optimum pH in the vicinity of neutrality.
* trade mark .
Regarding the optimum pH.of enzymes, it can be seen from the data presented in Figure 1 and Table II that the only glucoamylases which have their optimum pH's near the neutral zone are the present enzyme and the glucoamylase produced by the rice blast microorganism, Piricularia oryzae. However, as is clear from Figure 2 and Table. II, the present enzyme and the rice blast glucoamylase have optimum reaction temperatures which are extremely different.
In addition, the curves presented in Figure 4 indicate that the thermostability of the present enzyme is vastly superior to that of the rice blast glucoamylase Moreover, Table II
shows that the molecular weight of the present enzyme is much smaller than the molecular weight of the other kno~n glucoamylases.
TABLE II
COMPARISON OF VARIOUS GLUCOAMYLASES IN TERMS
OF OPTIMUM pH, OPTIMUM TEMPERATURE
AND MOLECULAR WEIGHT
Optimum Optimum Temp. Molecular Glucoamylase ~Ha) oCa~ Weighta) Present Enzyme 6.o-6.5 65 50,000 (Stachybotrys subsimplex) Rhizopus sp. (Sumyzyme)5.0 60 70,000b) Aspergillus niger 4.5* 70* 97,000C) Endomyces sp.d) 5.0 - 64,ooo Endomyces fibuligerae) 5.5 60 Trichoderma viride ) 5.0 60 75,000 Cephalosporium charticolag) 5.4 60 69,000 Piricular~a oryzaeh) 6.5 55 94,000 (rice blast org.
a) All values except those marked with an asterisk C~
were taken from the references.
b) Hiromi, et al: Biochem. Biophys. Acta 302, 362 (1973) c) J. H. Pazur, et al: J. Biol. Chem. 237, 1002 (1962).
d) Hattori, et al: Agr. Biol. Chem. 25 ~ 95 ~1961).
e) Harada, et al: J. Ferment. Tech. 53, 559 (1975).
f) Okada; J. Jap. Soc. Starch Sci. 21, 283 (1974).
g) H. Urbanek, et al: Appl. Micro. 30, 163 (1975).
h) Matsuda, et al: Amylase Symposium 2, 105 (1974).
On the basis of the above facts, it can be con-cluded that the glucoamylase produced by the method of the present invention is a new neutral glucoamylase which has been totally unknown to date.
An explanation will now be made of the method for the production of the present enzyme.
As a desirahle example of the glucoamylase-producing microorganism to be used in the present inven-tion, there is strain G30-1140, which was isolated from the soil by the present inventors. The identification of this strain will be presented first.
T~emorphological properties of tne present strain were determined in accordance with the methods described by the researchers listed below:
Gilman, J. C. A MANUAL OF SOIL FUNGI. The Iowa State University press, Ames. 1971.
Clements, F. E. and Shear, C. L. THE GENERA OF
FUNGI. Hafner, New York. 1964.
Barnett, H. L. ILLUSTRATED GENERA OF IMPERFECT
FUNGI. 2nd ed. Burgess, Minneapolis. 1968.
Z
.
3isby, G. R. Trans. Br l~lycol. Soc. 26, 133-43 (1943).
Ainsworth, G. C. DICTIONARY OF THE FUNGI.
6th ed. Commonwealth Mycological Institute, Kew, Surrey. 1971.
Next, the points of difference between the present enzyme and the conventionally-known glucoamylases will be presented, and an explanation will be made of the reasons that this enzyme is to be considered a new enzyme having its optimum pH in the vicinity of neutrality.
* trade mark .
Regarding the optimum pH.of enzymes, it can be seen from the data presented in Figure 1 and Table II that the only glucoamylases which have their optimum pH's near the neutral zone are the present enzyme and the glucoamylase produced by the rice blast microorganism, Piricularia oryzae. However, as is clear from Figure 2 and Table. II, the present enzyme and the rice blast glucoamylase have optimum reaction temperatures which are extremely different.
In addition, the curves presented in Figure 4 indicate that the thermostability of the present enzyme is vastly superior to that of the rice blast glucoamylase Moreover, Table II
shows that the molecular weight of the present enzyme is much smaller than the molecular weight of the other kno~n glucoamylases.
TABLE II
COMPARISON OF VARIOUS GLUCOAMYLASES IN TERMS
OF OPTIMUM pH, OPTIMUM TEMPERATURE
AND MOLECULAR WEIGHT
Optimum Optimum Temp. Molecular Glucoamylase ~Ha) oCa~ Weighta) Present Enzyme 6.o-6.5 65 50,000 (Stachybotrys subsimplex) Rhizopus sp. (Sumyzyme)5.0 60 70,000b) Aspergillus niger 4.5* 70* 97,000C) Endomyces sp.d) 5.0 - 64,ooo Endomyces fibuligerae) 5.5 60 Trichoderma viride ) 5.0 60 75,000 Cephalosporium charticolag) 5.4 60 69,000 Piricular~a oryzaeh) 6.5 55 94,000 (rice blast org.
a) All values except those marked with an asterisk C~
were taken from the references.
b) Hiromi, et al: Biochem. Biophys. Acta 302, 362 (1973) c) J. H. Pazur, et al: J. Biol. Chem. 237, 1002 (1962).
d) Hattori, et al: Agr. Biol. Chem. 25 ~ 95 ~1961).
e) Harada, et al: J. Ferment. Tech. 53, 559 (1975).
f) Okada; J. Jap. Soc. Starch Sci. 21, 283 (1974).
g) H. Urbanek, et al: Appl. Micro. 30, 163 (1975).
h) Matsuda, et al: Amylase Symposium 2, 105 (1974).
On the basis of the above facts, it can be con-cluded that the glucoamylase produced by the method of the present invention is a new neutral glucoamylase which has been totally unknown to date.
An explanation will now be made of the method for the production of the present enzyme.
As a desirahle example of the glucoamylase-producing microorganism to be used in the present inven-tion, there is strain G30-1140, which was isolated from the soil by the present inventors. The identification of this strain will be presented first.
T~emorphological properties of tne present strain were determined in accordance with the methods described by the researchers listed below:
Gilman, J. C. A MANUAL OF SOIL FUNGI. The Iowa State University press, Ames. 1971.
Clements, F. E. and Shear, C. L. THE GENERA OF
FUNGI. Hafner, New York. 1964.
Barnett, H. L. ILLUSTRATED GENERA OF IMPERFECT
FUNGI. 2nd ed. Burgess, Minneapolis. 1968.
Z
.
3isby, G. R. Trans. Br l~lycol. Soc. 26, 133-43 (1943).
Ainsworth, G. C. DICTIONARY OF THE FUNGI.
6th ed. Commonwealth Mycological Institute, Kew, Surrey. 1971.
9. Morphological Properties of Strain-G30-1140 The present strain was cultured on five kinds of media in Petri dishes. The following sections present the morphological characteristics which were observed for isolated colonies.
a) Czapek Agar Medium When incubated at 30C for 10 days, the colonies are thin and round, with a diameter of 4 to 5 cm. The vegetative hyphal are hyalin and show poor growth with hlack conidial clusters scattered like powder over the surface of the colonies. T~e undersides of the colonies are a brown color, and a tan pigment is secreted into the medium.
The vegetative hyphal consist of branched fibers which rarely possess any septa; the conidial structure is uniformly supported by the fibers. The conidiophores which have septa protrude from this at right angles. The length of the conidiophores is usually from 40 to 60 ~, but sometimes they attain more than 100 ~.
The diameter of these is about 3 to 6 ~, and although there are cases when the basal area of these is smooth, most of their surface is verrucose, being covered with fine granular projections. These conidiophores are hyalin, and most are not branched.
Z
On the apex of the cGnidiophores, hyalin phialides form whorls of 3 to 8 units. The shape of the ph~alides is ovoid or flask-like; they are 8 to 15 ~
in length and have a diameter of 2 to 6 ~; their surface is smooth. The conidia are formed at the apex of the phialides and are oval shapes of 3 to 5 ~ x 5 to 10 ~, and have a smooth surface. These are hyalin at the time of formation, but become blackish green as they mature.
The surface of the conidia is covered with a large amount of viscous material. For this reason, the conidia stick together and form large conidial clusters at the apex of the conidiophores. The viscous material is transparent at the time that it is formed, but then gradually becomes black.
b) Modified Czapek Agar Medium Percent Soluble Starch 1.0 Corn Steep Liquor (dry solids basis) 0.1 NaN03 0.2 K2HP0 4 - 0 . 1 KCl O 05 MgS04 7H20 ~ 0.05 FeS04 7H O 0.001 Agar 2.0 pH to 7.0 with NaOH
The growth of colonies on this medium is a bit slower than on the previously described Czapek medium, reaching about 3 cm when incubated for 10 days at 30C.
The colonies are circular and thin, and their surfaces have radiating from their centers a black viscous material which is in the form of oil-like drops having diameters reaching 1 to 3 mm. These arise from the gathering together of clusters of conidia which are enclosed in the viscous material and then form oil-drop-like bodies. The undersides of the colonies show a darker brown color than is seen with the previous Czapek medium, and a small amount of brown pigment is secreted into the medium.
c) Potato-Dex rose Agar Medium The growth of colonies on this medium is a bit slower than on the previously-described Czapek medium, reaching 3 to 4 cm when incubated for 10 days at 30C.
These colonies are also circular, but they have a somewhat greater thickness than the colonies on the Czapek medium. The growth of the vegetative cells is good, developing in a radiating pattern. The surfaces .
of the colonies are black with a slightly green luster, and are rich in hyphae, conidial clusters and so on.
After 14 days of incubation, the surfaces of the old colonies have radiating formations of synnemata standing about l to 3 mm erect. The undersides of these colonies show a blackish-brown color, and a large amount of brown pigment is secreted into the medium.
d) Special Agar Medium Percent Soluble Starch 1.0 Corn Steep Liquor (dry solids basis) 0.2 Cottonseed Oil Dregs 0.1 Yeast Extract 0.1 K2HP04 0.1 MgS04 7H20 0.05 - Agar 2.0 - pH to 7.0 with NaOH
- The colonies on this medium after 10 days of incubation ~t 30C have diameters of 5 to 6 cm, and are round and thin. The vegetative hyphae show good growth and have a black luster. The growth of Gonidia is worse than on the above-mentioned media. The undersides of the colonies are tan in color, and a tan pigment is released into the medium.
e) Davis's Yeast Salt Agar Medium The colonies on this medium after 10 days of incubation at 30C have diameters of 2 to 3 cm znd are more oval than round in shape. The hyphae are tan with ~13~Z
a touch of white and form somewhat thick colonles which are velvety. The undersides of the colonies are tan in color, but absolutely no pigment is secreted into the medium.
a) Czapek Agar Medium When incubated at 30C for 10 days, the colonies are thin and round, with a diameter of 4 to 5 cm. The vegetative hyphal are hyalin and show poor growth with hlack conidial clusters scattered like powder over the surface of the colonies. T~e undersides of the colonies are a brown color, and a tan pigment is secreted into the medium.
The vegetative hyphal consist of branched fibers which rarely possess any septa; the conidial structure is uniformly supported by the fibers. The conidiophores which have septa protrude from this at right angles. The length of the conidiophores is usually from 40 to 60 ~, but sometimes they attain more than 100 ~.
The diameter of these is about 3 to 6 ~, and although there are cases when the basal area of these is smooth, most of their surface is verrucose, being covered with fine granular projections. These conidiophores are hyalin, and most are not branched.
Z
On the apex of the cGnidiophores, hyalin phialides form whorls of 3 to 8 units. The shape of the ph~alides is ovoid or flask-like; they are 8 to 15 ~
in length and have a diameter of 2 to 6 ~; their surface is smooth. The conidia are formed at the apex of the phialides and are oval shapes of 3 to 5 ~ x 5 to 10 ~, and have a smooth surface. These are hyalin at the time of formation, but become blackish green as they mature.
The surface of the conidia is covered with a large amount of viscous material. For this reason, the conidia stick together and form large conidial clusters at the apex of the conidiophores. The viscous material is transparent at the time that it is formed, but then gradually becomes black.
b) Modified Czapek Agar Medium Percent Soluble Starch 1.0 Corn Steep Liquor (dry solids basis) 0.1 NaN03 0.2 K2HP0 4 - 0 . 1 KCl O 05 MgS04 7H20 ~ 0.05 FeS04 7H O 0.001 Agar 2.0 pH to 7.0 with NaOH
The growth of colonies on this medium is a bit slower than on the previously described Czapek medium, reaching about 3 cm when incubated for 10 days at 30C.
The colonies are circular and thin, and their surfaces have radiating from their centers a black viscous material which is in the form of oil-like drops having diameters reaching 1 to 3 mm. These arise from the gathering together of clusters of conidia which are enclosed in the viscous material and then form oil-drop-like bodies. The undersides of the colonies show a darker brown color than is seen with the previous Czapek medium, and a small amount of brown pigment is secreted into the medium.
c) Potato-Dex rose Agar Medium The growth of colonies on this medium is a bit slower than on the previously-described Czapek medium, reaching 3 to 4 cm when incubated for 10 days at 30C.
These colonies are also circular, but they have a somewhat greater thickness than the colonies on the Czapek medium. The growth of the vegetative cells is good, developing in a radiating pattern. The surfaces .
of the colonies are black with a slightly green luster, and are rich in hyphae, conidial clusters and so on.
After 14 days of incubation, the surfaces of the old colonies have radiating formations of synnemata standing about l to 3 mm erect. The undersides of these colonies show a blackish-brown color, and a large amount of brown pigment is secreted into the medium.
d) Special Agar Medium Percent Soluble Starch 1.0 Corn Steep Liquor (dry solids basis) 0.2 Cottonseed Oil Dregs 0.1 Yeast Extract 0.1 K2HP04 0.1 MgS04 7H20 0.05 - Agar 2.0 - pH to 7.0 with NaOH
- The colonies on this medium after 10 days of incubation ~t 30C have diameters of 5 to 6 cm, and are round and thin. The vegetative hyphae show good growth and have a black luster. The growth of Gonidia is worse than on the above-mentioned media. The undersides of the colonies are tan in color, and a tan pigment is released into the medium.
e) Davis's Yeast Salt Agar Medium The colonies on this medium after 10 days of incubation at 30C have diameters of 2 to 3 cm znd are more oval than round in shape. The hyphae are tan with ~13~Z
a touch of white and form somewhat thick colonles which are velvety. The undersides of the colonies are tan in color, but absolutely no pigment is secreted into the medium.
10. Physiological Properties of Strain G3~114Q
a) Growth Temperature This strain is capable of growth over a temperature range of 10 to 37C, but its optimum growth temperature is in the vicinity of 30C.
b) Growth pH
This strain is capable of growth over a pH
range o~ 3 to 10, but its optimum growth pH is in the vicinity of pH 7.
c) Carbon Source This strain i5 capable of using such carbon sources as dextrose, fructose, galactose, mannose, saccharose, maltose and starch in order to support its growth.
On the basis of the above microbiological findings, strain G30-1140 was identified as Gliobotrys alboviridis after consulting the GENERA OF FUNGI and A MANUAL OF SOIL FUNGI. However, according to the DICTIONARY OF THE FUNGI and G. R. Bisby (Trans. Br.
~L~llL~Z
Mycol. Soc. 26, 133-43 (1943)), this organism is the same as Stachybotrys subsimpleX, and for this reason strain G30-1140 was identified as Stachybotrys subsimplex.
Strain G30-1140 has conidiophores which stand erect from its vegetative hyphae, branching is almost nonexistent, and they have septa. There are occasions when the basal part is smooth, but the apex is covered with projections. At the apex,a level of phialides form a whorl of 3 to 8 units. Conidia having smooth oblong surfaces divide from these phialides, and they are enclosed in a richly viscous substance. These properties agree well with those described for Stachybotrys subsimplex by G. R. Bisby (Trans. Br. Mycol. Soc. 26, 133-43 (1943)).
This Stachybotrys subsimplex strain G30-1140 is being stored at the Fermentation Research Institute, Agency of Industrial Science & Technology, Chiba City, Japan, as Deposit No. 4377.
Regarding the cultivation of the microorganism to be employed in the present invention, the general knowledge and techniques used in the culture of molds are appllcable.
Namely, as the nutritional source medium, it is possible to employ the media which are used for the culture of ordinary molds. For example, various starches, starch hydrolyzates, corn meal, wheat flour, final molasses, etc., can be employed as carbon sources, while ~17-~ ~ 31~
the nitrogen requlrement can be supplied in the form of peptone, cottonseed oil dregs, meat extract, yeast extract, casein, corn steep liquor, malt extract, soybean dregs, skimmed milk~ inorganic ammonium sa~ts, inorganic nitrates, etc. As the inorganic salts, it is possible to employ calcium chloride, magnesium sulfate, phosphates~ sodium chloride, potassium chloride, and so on. Furthermore, these carbon sources, nitrogen sources and inorganic salts can be used either singly or in appropriate combinations. In addition, when it is desired to promote the growth of the microorganism and bring about an increase in its enzyme production, it is possible to employ trace amounts of metallic salts, vitamins, amino acids, and so forth.
~ The culture conditions usually employed for molds are also applicable to the cultivation of this microorganism. Namely, in liquid culture, if this mlcrobe is cultured for 7 to 14 days at pH 5 to 8 and 20C to 37C together with agitation to provide aeration, the enzyme of the present invention is accumulated in the culture fluid. In addition, if solid materials such as bran are employed, it is possible to carry out solid culture.
Next, an example will be presented of a method whereby the new neutral glucoamylase which is the objective of the present invention can be recovered from the cultured material. In the case of liquid culture, the mycelia are eliminated by any of the publicly-known methods; then the filtrate can be concentrated under reduced pressure, or the enzyme can be salted out with the other proteins by adding inorganic salts such as ammonium sulfate to the filtrate, or the enzyme can be precipitated out and concentrated by the addition of an organic solvent such as acetone or isopropanol.
.
In the case of solid culture~ the e.nzyme is first extracted from the. cultured material by the use of water or a buffer solution. Then, as in the case of liquid culture, it is possible to obtain the enzyme in a concentrated form.
The crude preparations of this new neutral enzyme obtained in this way can then be purified by carrying out the previously-mentioned purification techni~ues.
It is possible to employ this new neutral glucoamylase of the present invention for the saccharl-fication of liquefied starch when producing dextrose from starch. Especially, if the present enzyme is used and the saccharification is carried out at pH 6.o to 6.5, ~here is, as was mentioned earlier, little reverse reaction occurrence, and this results in an increased yield of dextrose being obtainable in comparison with the cases of employing the conventional glucoamylases and carrying out the saccharification under acidic conditions.
Z
The invention is further illustr~ted by reference to the following examples in which all parts and percentages are by weight unless otherwise noted.
A liquid culture medium containing 5% soluble starch, 2% corn steep liquor, 0.5% cottonseed oil dregs, 0.5% yeast extract~ 0.1% dipotassium phosphate, 0.05%
magnesium sulfate and 0.01% calcium chloride was adjusted to pH 7.0 and 100 ml of this was placed in a 500-ml Erlenmeyer flask. This medium was sterilized at 121C
for 10 minutes, inoculated with Stachybotrys subsimplex strain G30-1140, and incubated at 30C for 7 days on a shaker. After the culture was completed, the mycelia were eliminated from the culture fluid by filtration. The filtrate was found to contain 70 units of glucoamylase activity per milliliter.
This filtrate was next frozen for one night at -20C and then thawed at room temperature. The insoluble matter was removed by centrifugation. Two volumes of cold isopropanol was then added to this solution and it was left standing at 4C for one night so that the enzyme would be precipitated out. The supernatant was removed by decantation and the precipitate was dlssolved in a 0.05 M tris-HCl buffer solution containing 1 mM EDTA
and having a pH of 7.5. This enzyme-containing solution was then dialyzed against the same buf~er at 4~C for one night. DEAE-cellulose which had been equilibrated with the same buffer solution was then added to the dialyzed enzyme solution and the enzyme was adsorbed to this carrier. After washing this DEAE-dellulose with the same buffer, the enzyme was eluted from it using a solution of the same buffer containing NaCl at a con~
centration of 0.3 M. Next, two volumes of cold isopropanol was added to the eluate to cause the enzyme to precipitate, and the precipitate was collected by centrifugation. This precipitate was then dissolved in the 0.05 M tris-HCl buffer (pH 7.5j containing 1 mM
EDTA, followed by overnight dialysis against the same buffer solution. The dialyzed enzyme solution was next applied to a column of DEAE-cellulose which had been equilibrated with the same 0.05 M tris-HCl buffer (pH 7.5) containing 1 mM EDTA. Elution of the enzyme from this column was carried out by linearly increasing the con-centration of NaCl in the same buffer solution up to 0.5 M. The fractions of the eluate which contained the enzyme were then pooled and two volumes of cold iso-propanol was added in order to precipitate the enzyme out of this solution and concentrate it. The concentrated enzyme was next applied to a column of Sephadex G-150 which had been equilibrated with a 0.05 M tris-HCl buffer solution (pH 7.0) containing 1 mM EDTA, and elution was carried out using the same buffer. The eluted fractions 1~311~2 which showed enzyme ac~ ity were then pooled, and two ~olumes of cold isopropanol was added to this to preciPi-tate out the enzyme. This resulted in the recovery of the enzyme in a purified and concentrated form. The specific activity of this purified enzyme was found to be 127 units per milligram of protein.
To a 30% solution of a spray-dried maltodextrin (D.E. about 10) in 0.05 M acetate buffer at pH 6.5 was added the purified glucoamylase of Example 1. The enzyme was added at a dosage of 0.20 units of enzy~e per gram of substrate on a dry solids basis. After the solution had been incubated at 55C for 72 hours, the dextrose content of the filtered hydrolyzate,as determined by high performance liquid chromatography, was 96.5% of the total carbohydrate.
.
Starch was converted to a 10.2 D.E. starch hydrolyzate using bacterial alpha-amylase from B.
licheniformis according to the general procedure given in U.S. Patent 3,912,590. The solution was boiled for 5 minutes after adjusting the pH to 2.0 with 2 N HCl to inactivate the residual alpha-amylase. The starch hydrolyzate solution was then adjusted to pH 6.2 and diluted to the desired concentration before treatment with 0.20 units of the purified glucoamylase of Exam?le 1 per gram of substrate (dry solids basis). The solution ~3114Z
was incubated at 55C in a stoppered tube. The pH was ad~usted to 6.2 a~ter 5 hours and 48 hours. After the solution had been incubated for 72 hours, the dextrose content of the filtered hydrolyzate,as determined by high performance liquid chromatography, was 97.6p of the total carbohydrate. The final concentration of the solution was 31.2% on a dry solids basis.
When saccharification tests at the same substrate concentration were carried out with commercial glucoamylase from A. niger under its optimum conditions (pH 4.3 at 60C)7 - the corresponding dextrose yield was 96.5%. Similarly, the glucoamylase from R. niveus at pH 5.0 and 55C gave a dextrose yield of 97%. Dextrose yields were about 1~ lower when the saccharification tests were carried out with the commercial glucoamylases under the conditions used for the new enzyme. These results show that the new glucoamylase of this invention gives higher yields of dextrose than do the commercial glucoamylases even when each enzyme is utilized under its optimum reaction conditions.
a) Growth Temperature This strain is capable of growth over a temperature range of 10 to 37C, but its optimum growth temperature is in the vicinity of 30C.
b) Growth pH
This strain is capable of growth over a pH
range o~ 3 to 10, but its optimum growth pH is in the vicinity of pH 7.
c) Carbon Source This strain i5 capable of using such carbon sources as dextrose, fructose, galactose, mannose, saccharose, maltose and starch in order to support its growth.
On the basis of the above microbiological findings, strain G30-1140 was identified as Gliobotrys alboviridis after consulting the GENERA OF FUNGI and A MANUAL OF SOIL FUNGI. However, according to the DICTIONARY OF THE FUNGI and G. R. Bisby (Trans. Br.
~L~llL~Z
Mycol. Soc. 26, 133-43 (1943)), this organism is the same as Stachybotrys subsimpleX, and for this reason strain G30-1140 was identified as Stachybotrys subsimplex.
Strain G30-1140 has conidiophores which stand erect from its vegetative hyphae, branching is almost nonexistent, and they have septa. There are occasions when the basal part is smooth, but the apex is covered with projections. At the apex,a level of phialides form a whorl of 3 to 8 units. Conidia having smooth oblong surfaces divide from these phialides, and they are enclosed in a richly viscous substance. These properties agree well with those described for Stachybotrys subsimplex by G. R. Bisby (Trans. Br. Mycol. Soc. 26, 133-43 (1943)).
This Stachybotrys subsimplex strain G30-1140 is being stored at the Fermentation Research Institute, Agency of Industrial Science & Technology, Chiba City, Japan, as Deposit No. 4377.
Regarding the cultivation of the microorganism to be employed in the present invention, the general knowledge and techniques used in the culture of molds are appllcable.
Namely, as the nutritional source medium, it is possible to employ the media which are used for the culture of ordinary molds. For example, various starches, starch hydrolyzates, corn meal, wheat flour, final molasses, etc., can be employed as carbon sources, while ~17-~ ~ 31~
the nitrogen requlrement can be supplied in the form of peptone, cottonseed oil dregs, meat extract, yeast extract, casein, corn steep liquor, malt extract, soybean dregs, skimmed milk~ inorganic ammonium sa~ts, inorganic nitrates, etc. As the inorganic salts, it is possible to employ calcium chloride, magnesium sulfate, phosphates~ sodium chloride, potassium chloride, and so on. Furthermore, these carbon sources, nitrogen sources and inorganic salts can be used either singly or in appropriate combinations. In addition, when it is desired to promote the growth of the microorganism and bring about an increase in its enzyme production, it is possible to employ trace amounts of metallic salts, vitamins, amino acids, and so forth.
~ The culture conditions usually employed for molds are also applicable to the cultivation of this microorganism. Namely, in liquid culture, if this mlcrobe is cultured for 7 to 14 days at pH 5 to 8 and 20C to 37C together with agitation to provide aeration, the enzyme of the present invention is accumulated in the culture fluid. In addition, if solid materials such as bran are employed, it is possible to carry out solid culture.
Next, an example will be presented of a method whereby the new neutral glucoamylase which is the objective of the present invention can be recovered from the cultured material. In the case of liquid culture, the mycelia are eliminated by any of the publicly-known methods; then the filtrate can be concentrated under reduced pressure, or the enzyme can be salted out with the other proteins by adding inorganic salts such as ammonium sulfate to the filtrate, or the enzyme can be precipitated out and concentrated by the addition of an organic solvent such as acetone or isopropanol.
.
In the case of solid culture~ the e.nzyme is first extracted from the. cultured material by the use of water or a buffer solution. Then, as in the case of liquid culture, it is possible to obtain the enzyme in a concentrated form.
The crude preparations of this new neutral enzyme obtained in this way can then be purified by carrying out the previously-mentioned purification techni~ues.
It is possible to employ this new neutral glucoamylase of the present invention for the saccharl-fication of liquefied starch when producing dextrose from starch. Especially, if the present enzyme is used and the saccharification is carried out at pH 6.o to 6.5, ~here is, as was mentioned earlier, little reverse reaction occurrence, and this results in an increased yield of dextrose being obtainable in comparison with the cases of employing the conventional glucoamylases and carrying out the saccharification under acidic conditions.
Z
The invention is further illustr~ted by reference to the following examples in which all parts and percentages are by weight unless otherwise noted.
A liquid culture medium containing 5% soluble starch, 2% corn steep liquor, 0.5% cottonseed oil dregs, 0.5% yeast extract~ 0.1% dipotassium phosphate, 0.05%
magnesium sulfate and 0.01% calcium chloride was adjusted to pH 7.0 and 100 ml of this was placed in a 500-ml Erlenmeyer flask. This medium was sterilized at 121C
for 10 minutes, inoculated with Stachybotrys subsimplex strain G30-1140, and incubated at 30C for 7 days on a shaker. After the culture was completed, the mycelia were eliminated from the culture fluid by filtration. The filtrate was found to contain 70 units of glucoamylase activity per milliliter.
This filtrate was next frozen for one night at -20C and then thawed at room temperature. The insoluble matter was removed by centrifugation. Two volumes of cold isopropanol was then added to this solution and it was left standing at 4C for one night so that the enzyme would be precipitated out. The supernatant was removed by decantation and the precipitate was dlssolved in a 0.05 M tris-HCl buffer solution containing 1 mM EDTA
and having a pH of 7.5. This enzyme-containing solution was then dialyzed against the same buf~er at 4~C for one night. DEAE-cellulose which had been equilibrated with the same buffer solution was then added to the dialyzed enzyme solution and the enzyme was adsorbed to this carrier. After washing this DEAE-dellulose with the same buffer, the enzyme was eluted from it using a solution of the same buffer containing NaCl at a con~
centration of 0.3 M. Next, two volumes of cold isopropanol was added to the eluate to cause the enzyme to precipitate, and the precipitate was collected by centrifugation. This precipitate was then dissolved in the 0.05 M tris-HCl buffer (pH 7.5j containing 1 mM
EDTA, followed by overnight dialysis against the same buffer solution. The dialyzed enzyme solution was next applied to a column of DEAE-cellulose which had been equilibrated with the same 0.05 M tris-HCl buffer (pH 7.5) containing 1 mM EDTA. Elution of the enzyme from this column was carried out by linearly increasing the con-centration of NaCl in the same buffer solution up to 0.5 M. The fractions of the eluate which contained the enzyme were then pooled and two volumes of cold iso-propanol was added in order to precipitate the enzyme out of this solution and concentrate it. The concentrated enzyme was next applied to a column of Sephadex G-150 which had been equilibrated with a 0.05 M tris-HCl buffer solution (pH 7.0) containing 1 mM EDTA, and elution was carried out using the same buffer. The eluted fractions 1~311~2 which showed enzyme ac~ ity were then pooled, and two ~olumes of cold isopropanol was added to this to preciPi-tate out the enzyme. This resulted in the recovery of the enzyme in a purified and concentrated form. The specific activity of this purified enzyme was found to be 127 units per milligram of protein.
To a 30% solution of a spray-dried maltodextrin (D.E. about 10) in 0.05 M acetate buffer at pH 6.5 was added the purified glucoamylase of Example 1. The enzyme was added at a dosage of 0.20 units of enzy~e per gram of substrate on a dry solids basis. After the solution had been incubated at 55C for 72 hours, the dextrose content of the filtered hydrolyzate,as determined by high performance liquid chromatography, was 96.5% of the total carbohydrate.
.
Starch was converted to a 10.2 D.E. starch hydrolyzate using bacterial alpha-amylase from B.
licheniformis according to the general procedure given in U.S. Patent 3,912,590. The solution was boiled for 5 minutes after adjusting the pH to 2.0 with 2 N HCl to inactivate the residual alpha-amylase. The starch hydrolyzate solution was then adjusted to pH 6.2 and diluted to the desired concentration before treatment with 0.20 units of the purified glucoamylase of Exam?le 1 per gram of substrate (dry solids basis). The solution ~3114Z
was incubated at 55C in a stoppered tube. The pH was ad~usted to 6.2 a~ter 5 hours and 48 hours. After the solution had been incubated for 72 hours, the dextrose content of the filtered hydrolyzate,as determined by high performance liquid chromatography, was 97.6p of the total carbohydrate. The final concentration of the solution was 31.2% on a dry solids basis.
When saccharification tests at the same substrate concentration were carried out with commercial glucoamylase from A. niger under its optimum conditions (pH 4.3 at 60C)7 - the corresponding dextrose yield was 96.5%. Similarly, the glucoamylase from R. niveus at pH 5.0 and 55C gave a dextrose yield of 97%. Dextrose yields were about 1~ lower when the saccharification tests were carried out with the commercial glucoamylases under the conditions used for the new enzyme. These results show that the new glucoamylase of this invention gives higher yields of dextrose than do the commercial glucoamylases even when each enzyme is utilized under its optimum reaction conditions.
Claims (7)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A glucoamylase enzyme preparation,prepared by culturing cells of a strain of Stachybotrys subsimplex in a nutrient medium and isolating the glucoamylase enzyme preparation from the culture medium, which comprises a glucoamylase with a molecular weight of about 50,000 as determined by Sephadex G-150 column chromatography.
2. The enzyme preparation of claim 1 wherein the strain of Stachybotrys simplex is Fermentation Research Institute Deposit No. 4377.
3. The glucoamylase enzyme preparation of claim 1 which has an optimum glucoamylase activity in the range of about pH 6.0 to about 6.5 at 60°C and a maximum glucoamylase activity at about 65°C as measured by a 10-minute reaction on a 2% maltodextrin solution at pH 6Ø
4. The glucoamylase enzyme preparation of claim 1 which is capable of converting a 30% by weight solution of a 10 D.E.
starch hydrolyzate to a product containing at least about 96%
dextrose on a dry solids basis when reacted with the starch hydrolyzate at pH 6.0 to 6.5 at 55°C.
starch hydrolyzate to a product containing at least about 96%
dextrose on a dry solids basis when reacted with the starch hydrolyzate at pH 6.0 to 6.5 at 55°C.
5. In a process for producing a syrup of high dextrose content by saccharifying a liquefied starch to dextrose, the improvement which comprises saccharifying the liquefied starch at pH between 6.0 and 6.5 in the presence of the glucoamylase enzyme preparation of claim 1.
6. The process of claim 5 wherein the glucoamylase is obtained from the strain of Stachybotrys subsimplex, Fermentation Research Institute Deposit No. 4377.
7. The process of claim 6 wherein the saccharification is carried out at a temperature of from about 50°C to about 65°C.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8781278A JPS5515720A (en) | 1978-07-20 | 1978-07-20 | Industrially usable novel heat resistant neutral glucoamylase and method |
JP53-87812 | 1978-07-20 | ||
US055,717 | 1979-07-09 | ||
US06/055,717 US4254225A (en) | 1978-07-20 | 1979-07-09 | Novel neutral glucoamylase and method for its production |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1131142A true CA1131142A (en) | 1982-09-07 |
Family
ID=26429060
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA332,250A Expired CA1131142A (en) | 1978-07-20 | 1979-07-20 | Glucoamylase from stachybotrys subsimplex |
Country Status (9)
Country | Link |
---|---|
AR (1) | AR225420A1 (en) |
AU (1) | AU527668B2 (en) |
CA (1) | CA1131142A (en) |
DK (1) | DK146941C (en) |
ES (2) | ES482625A1 (en) |
GB (1) | GB2025978B (en) |
IT (1) | IT1193794B (en) |
MX (1) | MX6277E (en) |
MY (1) | MY8400139A (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4536477A (en) * | 1983-08-17 | 1985-08-20 | Cpc International Inc. | Thermostable glucoamylase and method for its production |
ATE95837T1 (en) * | 1984-08-06 | 1993-10-15 | Genencor Inc | ENZYMATIC HYDROLYSIS OF GRANULAR STARCH DIRECTLY TO GLUCOSE. |
-
1979
- 1979-07-17 IT IT7924432A patent/IT1193794B/en active
- 1979-07-18 AR AR277356A patent/AR225420A1/en active
- 1979-07-18 AU AU49044/79A patent/AU527668B2/en not_active Ceased
- 1979-07-18 GB GB7925069A patent/GB2025978B/en not_active Expired
- 1979-07-19 ES ES482625A patent/ES482625A1/en not_active Expired
- 1979-07-19 DK DK303379A patent/DK146941C/en not_active IP Right Cessation
- 1979-07-19 MX MX798207U patent/MX6277E/en unknown
- 1979-07-19 ES ES482619A patent/ES482619A1/en not_active Expired
- 1979-07-20 CA CA332,250A patent/CA1131142A/en not_active Expired
-
1984
- 1984-12-30 MY MY139/84A patent/MY8400139A/en unknown
Also Published As
Publication number | Publication date |
---|---|
MY8400139A (en) | 1984-12-31 |
GB2025978A (en) | 1980-01-30 |
AU4904479A (en) | 1980-01-24 |
IT1193794B (en) | 1988-08-24 |
DK146941C (en) | 1984-07-30 |
AU527668B2 (en) | 1983-03-17 |
IT7924432A0 (en) | 1979-07-17 |
MX6277E (en) | 1985-03-05 |
DK146941B (en) | 1984-02-20 |
ES482625A1 (en) | 1980-04-16 |
GB2025978B (en) | 1982-12-22 |
AR225420A1 (en) | 1982-03-31 |
ES482619A1 (en) | 1980-04-16 |
DK303379A (en) | 1980-01-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA1128885A (en) | Thermostable glucoamylase from talaromyces duponti | |
US4727026A (en) | Method for direct saccharification of raw starch using enzyme produced by a basidiomycete belonging to the genus Corticium | |
US4284722A (en) | Heat and acid-stable alpha-amylase enzymes and processes for producing the same | |
US4591560A (en) | Process for saccharification of starch using enzyme produced by fungus belonging to genus Chalara | |
EP0063909A1 (en) | Debranching enzyme product, preparation and use thereof | |
CA1262879A (en) | PROCESS FOR PRODUCING THERMOSTABLE .alpha.-AMYLASES BY CULTURING MICRO-ORGANISMS AT ELEVATED TEMPERATURES | |
EP0184019B1 (en) | Thermostable alpha-amylase-producing, thermophilic anaerobic bacteria, thermostable alpha-amylase and process for producing the same | |
US4211842A (en) | Starch-degrading benzymes derived from Clacosporium resinae | |
Michelena et al. | Production of amylase by Aspergillus foetidus on rice flour medium and characterization of the enzyme | |
Tani et al. | Raw cassava starch-digestive glucoamylase of Aspergillus sp. N-2 isolated from cassava chips | |
EP0138428A2 (en) | Acid-stable alpha-amylase composition, preparation and use thereof | |
CA1131142A (en) | Glucoamylase from stachybotrys subsimplex | |
US4254225A (en) | Novel neutral glucoamylase and method for its production | |
CA1081633A (en) | Heat and acid-stable alpha-amylase enzymes and processes for producing the same | |
US4458017A (en) | Process for preparing fructose from starch | |
US4420562A (en) | Method for producing creatinase | |
KR100261359B1 (en) | Debranching enzyme and process for producing the same | |
JP3761236B2 (en) | Novel β-glucosidase, production method and use thereof | |
US4605619A (en) | Process for preparing fructose from starch | |
CA1195275A (en) | Process for preparing fructose | |
KR830002800B1 (en) | Preparation of heat stable glucoamylase | |
JPS6243671B2 (en) | ||
KR830002799B1 (en) | Preparation of novel neutral glucoamylases and their products | |
EP0528612A2 (en) | Amylase capable of digesting raw starch | |
JPH0313876B2 (en) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
MKEX | Expiry |