JPH0428356B2 - - Google Patents
Info
- Publication number
- JPH0428356B2 JPH0428356B2 JP61185129A JP18512986A JPH0428356B2 JP H0428356 B2 JPH0428356 B2 JP H0428356B2 JP 61185129 A JP61185129 A JP 61185129A JP 18512986 A JP18512986 A JP 18512986A JP H0428356 B2 JPH0428356 B2 JP H0428356B2
- Authority
- JP
- Japan
- Prior art keywords
- amylase
- starch
- glucoamylase
- activity
- liquid
- 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 - Lifetime
Links
- 108090000637 alpha-Amylases Proteins 0.000 claims description 93
- 102000004139 alpha-Amylases Human genes 0.000 claims description 93
- 229940024171 alpha-amylase Drugs 0.000 claims description 87
- 229920002472 Starch Polymers 0.000 claims description 75
- 235000019698 starch Nutrition 0.000 claims description 75
- 239000008107 starch Substances 0.000 claims description 69
- 102100022624 Glucoamylase Human genes 0.000 claims description 61
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims description 59
- 241000894006 Bacteria Species 0.000 claims description 23
- 229920001353 Dextrin Polymers 0.000 claims description 23
- 239000004375 Dextrin Substances 0.000 claims description 23
- 230000002378 acidificating effect Effects 0.000 claims description 23
- 235000019425 dextrin Nutrition 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 23
- 238000004519 manufacturing process Methods 0.000 claims description 21
- 239000002994 raw material Substances 0.000 claims description 17
- 241000193403 Clostridium Species 0.000 claims description 12
- 229910021645 metal ion Inorganic materials 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims 2
- 230000000694 effects Effects 0.000 description 69
- 239000007788 liquid Substances 0.000 description 49
- 239000000243 solution Substances 0.000 description 43
- 238000006243 chemical reaction Methods 0.000 description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 33
- 239000002609 medium Substances 0.000 description 29
- 102000004190 Enzymes Human genes 0.000 description 28
- 108090000790 Enzymes Proteins 0.000 description 28
- 229940088598 enzyme Drugs 0.000 description 28
- 239000001888 Peptone Substances 0.000 description 26
- 108010080698 Peptones Proteins 0.000 description 25
- 235000019319 peptone Nutrition 0.000 description 25
- 150000003839 salts Chemical class 0.000 description 22
- 230000012010 growth Effects 0.000 description 21
- 235000013372 meat Nutrition 0.000 description 21
- 229920001817 Agar Polymers 0.000 description 19
- 239000008272 agar Substances 0.000 description 19
- 239000002002 slurry Substances 0.000 description 19
- 238000003860 storage Methods 0.000 description 17
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 15
- 239000007789 gas Substances 0.000 description 14
- 239000000203 mixture Substances 0.000 description 14
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 13
- 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 description 13
- 239000002253 acid Substances 0.000 description 13
- 239000011575 calcium Substances 0.000 description 13
- 229910052791 calcium Inorganic materials 0.000 description 13
- 239000008103 glucose Substances 0.000 description 13
- 238000010438 heat treatment Methods 0.000 description 13
- GNBVPFITFYNRCN-UHFFFAOYSA-M sodium thioglycolate Chemical compound [Na+].[O-]C(=O)CS GNBVPFITFYNRCN-UHFFFAOYSA-M 0.000 description 13
- 229940046307 sodium thioglycolate Drugs 0.000 description 13
- 239000000758 substrate Substances 0.000 description 12
- 241000193830 Bacillus <bacterium> Species 0.000 description 11
- 239000000872 buffer Substances 0.000 description 11
- 239000012153 distilled water Substances 0.000 description 11
- 229910052751 metal Inorganic materials 0.000 description 11
- 239000002184 metal Substances 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 9
- 229910001424 calcium ion Inorganic materials 0.000 description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 9
- 238000010586 diagram Methods 0.000 description 9
- 238000005259 measurement Methods 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- 229920002261 Corn starch Polymers 0.000 description 8
- 230000009471 action Effects 0.000 description 8
- 239000008120 corn starch Substances 0.000 description 8
- 229940099112 cornstarch Drugs 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 8
- 239000008399 tap water Substances 0.000 description 8
- 235000020679 tap water Nutrition 0.000 description 8
- 108010010803 Gelatin Proteins 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000008273 gelatin Substances 0.000 description 7
- 229920000159 gelatin Polymers 0.000 description 7
- 235000019322 gelatine Nutrition 0.000 description 7
- 235000011852 gelatine desserts Nutrition 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 229940041514 candida albicans extract Drugs 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 238000006911 enzymatic reaction Methods 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 238000009630 liquid culture Methods 0.000 description 6
- 239000002808 molecular sieve Substances 0.000 description 6
- 238000010979 pH adjustment Methods 0.000 description 6
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 239000012138 yeast extract Substances 0.000 description 6
- 244000063299 Bacillus subtilis Species 0.000 description 5
- 235000014469 Bacillus subtilis Nutrition 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 239000007979 citrate buffer Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 239000000049 pigment Substances 0.000 description 5
- 229920001592 potato starch Polymers 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 239000004382 Amylase Substances 0.000 description 4
- 102000013142 Amylases Human genes 0.000 description 4
- 108010065511 Amylases Proteins 0.000 description 4
- 229920000856 Amylose Polymers 0.000 description 4
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 4
- 244000061456 Solanum tuberosum Species 0.000 description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 description 4
- 235000019418 amylase Nutrition 0.000 description 4
- 239000012298 atmosphere Substances 0.000 description 4
- 238000011033 desalting Methods 0.000 description 4
- 238000004255 ion exchange chromatography Methods 0.000 description 4
- 239000013028 medium composition Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 230000001681 protective effect Effects 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 229910052708 sodium Inorganic materials 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 3
- 244000017020 Ipomoea batatas Species 0.000 description 3
- 235000002678 Ipomoea batatas Nutrition 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 150000003863 ammonium salts Chemical class 0.000 description 3
- 159000000007 calcium salts Chemical class 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 235000005822 corn Nutrition 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 3
- 230000003301 hydrolyzing effect Effects 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000005374 membrane filtration Methods 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000005185 salting out Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 3
- 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 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical group [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- 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 2
- 108050008938 Glucoamylases Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 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
- VEQPNABPJHWNSG-UHFFFAOYSA-N Nickel(2+) Chemical compound [Ni+2] VEQPNABPJHWNSG-UHFFFAOYSA-N 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 108010046334 Urease Proteins 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 229940061607 dibasic sodium phosphate Drugs 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000001641 gel filtration chromatography Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 239000002198 insoluble material Substances 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 229940057995 liquid paraffin Drugs 0.000 description 2
- 229910001425 magnesium ion Inorganic materials 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000010755 mineral Nutrition 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 229910001453 nickel ion Inorganic materials 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- 239000003002 pH adjusting agent Substances 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 239000007974 sodium acetate buffer Substances 0.000 description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000003828 vacuum filtration Methods 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 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 1
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 240000004160 Capsicum annuum Species 0.000 description 1
- 235000008534 Capsicum annuum var annuum Nutrition 0.000 description 1
- 241001112696 Clostridia Species 0.000 description 1
- 241000193464 Clostridium sp. Species 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 229920002245 Dextrose equivalent Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000228341 Talaromyces Species 0.000 description 1
- 241000223258 Thermomyces lanuginosus Species 0.000 description 1
- 241000589596 Thermus Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- WJGAPUXHSQQWQF-UHFFFAOYSA-N acetic acid;hydrochloride Chemical compound Cl.CC(O)=O WJGAPUXHSQQWQF-UHFFFAOYSA-N 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- ABIUHPWEYMSGSR-UHFFFAOYSA-N bromocresol purple Chemical compound BrC1=C(O)C(C)=CC(C2(C3=CC=CC=C3S(=O)(=O)O2)C=2C=C(Br)C(O)=C(C)C=2)=C1 ABIUHPWEYMSGSR-UHFFFAOYSA-N 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 229910001429 cobalt ion Inorganic materials 0.000 description 1
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000021438 curry Nutrition 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000009229 glucose formation Effects 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 150000004688 heptahydrates Chemical class 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000008235 industrial water Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- -1 iron ions Chemical class 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- QSOMFNQEXNFPNU-UHFFFAOYSA-L magnesium;hydrogen sulfate;hydroxide;hydrate Chemical compound O.O.[Mg+2].[O-]S([O-])(=O)=O QSOMFNQEXNFPNU-UHFFFAOYSA-L 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 229910001437 manganese ion Inorganic materials 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000010815 organic waste Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- KVMLCRQYXDYXDX-UHFFFAOYSA-M potassium;chloride;hydrochloride Chemical compound Cl.[Cl-].[K+] KVMLCRQYXDYXDX-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical group O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000019086 sulfide ion homeostasis Effects 0.000 description 1
- 239000008400 supply water Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 235000013343 vitamin 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
- 238000005406 washing Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
〔産業上の利用分野〕
本発明は殿粉の糖化方法に係り、特に殿粉をぶ
どう糖に加水分解するのに好適な糖の製造方法及
びその製造プラントに関する。
〔従来の技術〕
殿粉からぶどう糖や異性化糖等の甘味料を製造
するには、殿粉をまずα−アミラーゼによりデキ
ストリンに加水分解し、しかるのちグルコアミラ
ーゼで異性化している（化学便覧応用編改訂２
版）。
このように、α−アミラーゼとグルコアミラー
ゼは殿粉加工工業において中心的役割をもつ酵素
である。
通常の酸素は常温以下でしか安定でないが、糖
製造ライン内の雑菌繁殖や反応の高速化のため60
℃以上のより高い温度域で反応させることが望ま
れてきた。さらに、原料の殿粉スラリは酸性の不
純物を含むため、PH４〜５の酸性を呈する。この
ため、酸性下で耐熱性を有する酸素が望まれてき
た。
現用のα−アミラーゼは耐熱性を有するが、反
応の最適PHが中性域にあり、かつ安定域も中性で
ある他、数mMのカルシウムイオンの共存下では
じめて耐熱性を発揮できる。先に、我々は、耐熱
性，耐酸性を有し、カルシウム剤の添加を必要と
しない新型のα−アミラーゼを見い出している。
このα−アミラーゼを用いれば、液化工程で酸性
のままカルシウムイオン無添加で、高温で反応さ
せることができる。
一方、グルコアミラーゼは前述したように、液
化反応に引きつづきデキストリンをぶどう糖に加
水分解する際用いられる。現在公知のグルコアミ
ラーゼは酸性下では耐熱性を有するものは知られ
ていない。
このため、液化反応で、耐酸，耐熱性α−アミ
ラーゼを用い、酸性のまま高温で反応させても糖
化工程でいつたん中和して反応しなければならな
い。したがつて、中和工程が必要となるだけでな
く、かつ糖製品化の際に行うイオン交換樹脂によ
る脱塩工程に大きな負担となる。
〔発明が解決しようとする問題点〕
本発明の目的は、上述の従来技術の欠点を解決
し、でん粉を原料としてぶどう糖を製造するにあ
たり特に、PH４〜５の酸性条件下、60〜70℃の高
温下で作用可能なグルコアミラーゼを糖化酵素と
して用いることにより冷却及び中和工程が不用な
糖の製造方法を提供することにある。
〔問題点を解決するための手段〕
本発明は、α−アミラーゼを用いて殿粉を液化
した後、グルコアミラーゼを用いて生成したデキ
ストリンを糖化する殿粉の糖化方法において、原
料殿粉を酸性で、かつ実質的に金属イオンの添加
なしに、70℃以上の温度で反応させて液化及び糖
化することにあり、具体的には上記のα−アミラ
ーゼとしては、作用最適PHが２〜5.5，PH4.5にお
ける80℃での活性半減期が80時間以上の耐酸性及
び耐熱性を有し、耐熱性発揮に10μM以下のカル
シウムイオンで活性を有する耐熱性α−アミラー
ゼを、またグルコアミラーゼとしては、作用最適
PHが４〜５，PH4.5における70℃での活性半減期
が３時間以上、65℃での活性半減期が120時間以
上の耐酸性及び耐熱性を有し、耐熱性発揮にカル
シウムイオンを必要としない耐熱性グルコアミラ
ーゼを用いることにある。かかるα−アミラーゼ
はクロスツリジウム属に属する微工研菌寄第7918
号の好熱性嫌気性細菌より産生することができ、
グルコアミラーゼはクロスツリジウム属に属する
微工研菌寄第8737号の好熱性嫌気性細菌より産生
することができる。
本発明者らは上記した従来の糖化方法の欠点を
改善するため、酸性下で高い耐熱性を有する新し
いグルコアミラーゼを得ることを目指し、酵素生
産用微生物の探索を行つた。
その結果、有機廃棄物の中温メタン発酵スラリ
中から分離した中等度好熱嫌気性細菌Ｇ−00003
が上記の条件を満たす新規な耐熱性グルコアミラ
ーゼを生成することを見い出した。そして、本菌
の培養液から本酵素を分離し、これを用いて殿粉
の糖化方法について鋭意検討した結果、本発明に
至つた。
本発明の第１の特徴は、本発明に係る新規耐熱
性グルコアミラーゼを用い、カルシウム剤無添加
で、かつ原料殿粉液を中和することなく酸性のま
ま糖化することである。
第２の特徴は、耐酸性，耐酸性，低カルシウム
要求性のα−アミラーゼを用いて液化した液化処
理液を、耐酸性，耐熱性を有するグルコアミラー
ゼで糖化することである。
したがつて、実質的に中和せずにかつ高速に糖
化できることとなり、糖化後の脱塩工程への負荷
が大巾に軽減されると共に、糖製造ラインの雑菌
繁殖も防止できる。
本発明の第３の特徴は、耐熱性，耐酸性，低カ
ルシウム要求性を有するα−アミラーゼと本発明
に係るグルコアミラーゼの同時存在下で殿粉を液
化，糖化することである。
本発明に使用できる、ぶどう糖の出発原料とな
る殿粉の種類は、特に限定されるものではなく、
例えば、馬鈴薯，甘しよ，とうもろこし，タピオ
キ，小麦などの公知の殿粉に十分適用できる。殿
粉の液化方法もα−アミラーゼを用いる酵素法で
も、化学的酸加水分解法であつてもよい。
本発明に係るα−アミラーゼの耐熱性保持を目
的とするカルシウム剤の添加は、仕込水に蒸溜水
を用い、かつ完全に脱カルシウム処理した試薬級
のアミロースを用いる場合を除外すれば必要な
い。したがつて、通常の水道水を用いれば、後に
行う陽イオン除去を目的とする脱塩工程は事実上
必要ない。液化に際してのPH調整は、本α−アミ
ラーゼの最適反応域及び安定域とほぼ一致してい
るため通常は必要ない。このため、酸性の殿粉ス
ラリーをPHの調整をせずに反応できる。
原料殿粉濃度は、原料の種類，品質，液化処理
液の用途によりことなるが、従来公知の10〜40％
の範囲内にある。
反応温度は、本α−アミラーゼの耐熱性から
100℃以下で用いられる。酵素の反応速度と耐熱
性を考慮すれば、70〜80℃で行うのが好ましい。
酵素の添加量は、反応槽内における原料殿粉の種
類及び滞留時間，反応温度，PH等により異なる
が、処理時間を0.5時間に固定した場合、80℃，
PH4.5にて馬鈴薯殿粉を限界デキストリンにまで
加水分解するには殿粉１Kgあたり約105単位を必
要とする。ここで、α−アミラーゼ活性の単位は
Blue value法によるもので、酵素の特性の個所
で詳述する。
本発明に使用できる殿粉液化処理物すなわちデ
キストランは、酵素法による液化処理によつても
化学的な酸部分分解処理によるものでもよい。
糖化に際してのPHの調整は、液化処理の際、PH
４〜５の酸性で行われる限り、通常は必要ない。
液化処理液の濃度は、原料の種類，品質，用途に
よりことなるが、従来公知の10〜40％の範囲内に
ある。
反応温度は、本発明に係るグルコアミラーゼの
耐熱性から100℃以下で行われる。酵素の反応速
度と耐熱性を考慮すれば60〜80℃で行うのが好ま
しい。
カルシウムイオンの添加は本酵素の特性からし
て不要である。
酵素の添加量は、反応槽内における原料液化処
理液の質や濃度，滞留時間，反応温度，PH等によ
り異なるが、処理時間を70℃，PH4.5にて限界デ
キストリンを糖化率90％まで糖化するには、局方
馬鈴薯殿粉１Kgあたり、約120単位を必要とする。
ここで、グルコアミラーゼの活性単位の定義に
ついては、酵素の特性の個所で詳述する。
本α−アミラーゼと本グルコアミラーゼの同時
存在下で液化，糖化を行う場合には、温度，PH等
は両酵素で同一範囲内にあることから、両酵素の
特性を勘案して適宜選定した条件下で行う。
本発明に用いるα−アミラーゼを産生するクロ
スツリジウム属に属する細菌（Clostridium sp
RS−0001）は工業技術院微生物工業技術研究所
に寄託している（受託番号；微工研菌寄第7918号
（FERM Ｐ−7918））である。まず、本菌の菌学
的性質の詳細を説明する。
Ａ 形態的性質
(1) 栄養細胞の形態
下記の殿粉・ペプトン培地の寒天平板上、嫌気
性範囲気中、60℃で２日間培養した場合、栄養細
胞は0.4〜0.8×２〜5μｍの大きさの直状の桿菌で
ある。３日間以上の培養では、上記の形状の栄養
細胞が単独に存在する他、連鎖するものも生ず
る。液体培養でも同様となる。
殿粉・ペプトン培地の組成
可溶性殿粉 1.5％
ペプトン 0.5％
酵母エキス 0.5％
KH2PO4 0.7％
Na2HPO4 0.35％
MgSO4・7H2Ｏ 0.001％
寒 天 2.0％
チオグリコール酸ナトリウム 0.1％
水道水
PH6.4
(2) 胞子の有無
殿粉・ペプトン培地の寒天平板培養及び液体培
養で胞子の形成が認められる。
Ｂ 培養的特性
(1) コロニーの形態
殿粉・ペプトン培地の寒天平板培養でのコロニ
ーは、中心部がやや隆起した扁平な円形となり、
周縁部は全縁である。色素生成は見られず、表面
に光沢を有し乳白色不透明である。また、粘着性
を有する。
(2) 肉汁培地の寒天平板培養及び穿刺絶養生育し
て殿粉・ペプトン培地と同様のコロニーを生ず
る。
肉汁寒天培地組成
肉エキス 1.0％
ペプトン 1.0％
食塩 0.2％
チオグリコール酸ナトリウム 0.1％
寒 天 1.5％
蒸留水
PH6.0
(3) 肉汁培地の穿刺培養
水素と炭酸ガスを含むガスの発生を伴つて生育
し、このため寒天培地が２〜３個所で分断され
る。
(4) 肉汁液体培養
嫌気的範囲気下でのみ生育し、培養液が自濁す
る。
肉汁培地の組成
肉エキス 1.0％
チオグリコール酸ナトリウム 0.1％
蒸留水
PH6.0
(5) 肉汁・ゼラチン培養
生育は認められない。
肉汁・ゼラチン培地の組成
肉エキス 1.0％
ペプトン 1.0％
食 塩 0.2％
ゼラチン 15％
チオグリコール酸ナトリウム 0.1％
蒸留水
PH6.0
(3) リトマスミルク培養
ガス発生を伴い、固く凝固し、酸の生成により
赤変する。
Ｃ 生理的性質
(1) 生育の温度範囲
40〜63℃で生育する。30℃では生育は認められ
ず、60℃付近で良好。
(2) 生育のPH範囲
PH５〜７。5.6付近が良好。
(3) 酸素に対する態度
偏性嫌気性
(4) Ｏ−Ｆテスト（Hugh Laifson変法）
空気範囲気中では生育みられず陰性。流動パラ
フイン重層による嫌気性条件下では菌が生育し、
酸を生成して培養液が黄色となる。
培地組成
ペプトン 0.2％
グルコース 1.0％
食 塩 0.5％
K2HPO4 0.03％
チオグリコール酸ナトリウム 0.1％
ブロムクレゾールパープル 0.002％
寒 天 0.3％
蒸留水
PH6.0
陰性。
(5) VPテスト
陰性。
(6) MRテスト
陽極，赤変化する。
(7) インドール生成
ペプトン水に生育しないため測定できない。
(8) 硫化水素の生成
陰性（Kligrerの培地使用において）。
(9) 殿粉の加水分解
陽性。可溶性殿粉だけでなく、馬鈴薯殿粉など
粒状殿粉も分解する。
(10) クエン酸の利用
陰性（Simmons培地使用において）。
(11) アンモニウム塩の利用
ペプトン水に生育しないため測定できない。
(12) 色素の菌体外生成
陰性。
(13) ウレアーゼ
陰性。
(14) オキシダーゼ活性
陰性。
(15) カタラーゼ活性
陰性。
(16) 糖の資化性
糖の資化性及びダラーム管を用いたガス発生有
無の観察結果を下表に示す。
[Industrial Field of Application] The present invention relates to a method for saccharification of starch, and particularly to a method for producing sugar suitable for hydrolyzing starch into glucose and a plant for producing the same. [Conventional technology] To produce sweeteners such as glucose and high-fructose sugar from starch, the starch is first hydrolyzed into dextrin with α-amylase, and then isomerized with glucoamylase (Chemistry Handbook Applications). Edited revision 2
edition). Thus, α-amylase and glucoamylase are enzymes that play a central role in the starch processing industry. Normal oxygen is only stable at temperatures below room temperature, but due to the proliferation of bacteria and faster reactions in sugar production lines,
It has been desired to carry out the reaction at a higher temperature range of ℃ or higher. Furthermore, the starch slurry used as a raw material contains acidic impurities, so it exhibits acidity with a pH of 4 to 5. For this reason, oxygen that has heat resistance under acidic conditions has been desired. Current α-amylases are thermostable, but the optimum pH for the reaction is in the neutral range, the stability range is also neutral, and they can only exhibit heat resistance in the coexistence of several mM calcium ions. Previously, we have discovered a new type of α-amylase that has heat resistance and acid resistance, and does not require the addition of calcium agents.
If this α-amylase is used, the reaction can be carried out at high temperature without adding calcium ions while remaining acidic in the liquefaction process. On the other hand, as mentioned above, glucoamylase is used when hydrolyzing dextrin into glucose following the liquefaction reaction. None of the currently known glucoamylases is known to have heat resistance under acidic conditions. Therefore, in the liquefaction reaction, acid-resistant and heat-stable α-amylase is used, and even if the reaction is carried out at high temperature while being acidic, it must be neutralized in the saccharification step before the reaction can proceed. Therefore, not only is a neutralization step required, but it also places a heavy burden on the desalting step using an ion exchange resin that is performed when producing sugar products. [Problems to be Solved by the Invention] The purpose of the present invention is to solve the above-mentioned drawbacks of the prior art, and particularly to produce glucose using starch as a raw material, under acidic conditions of pH 4 to 5 and at 60 to 70°C. The object of the present invention is to provide a method for producing sugar that does not require cooling and neutralization steps by using glucoamylase that can act at high temperatures as a saccharifying enzyme. [Means for Solving the Problems] The present invention provides a starch saccharification method in which starch is liquefied using α-amylase and then dextrin produced using glucoamylase is saccharified. The purpose is to liquefy and saccharify the α-amylase by reacting at a temperature of 70°C or higher without substantially adding metal ions. Thermostable α-amylase has acid resistance and heat resistance with a half-life of activity at 80℃ of 80 hours or more at pH 4.5, and is active with calcium ions of 10 μM or less to exert heat resistance, and as a glucoamylase. , action optimal
It has acid resistance and heat resistance with an active half-life of 3 hours or more at 70℃ and 120 hours or more at 65℃ at a pH of 4 to 5 and PH4.5, and calcium ions are used to exhibit heat resistance. The purpose is to use thermostable glucoamylase, which is not required. Such α-amylase belongs to the genus Clostridium.
It can be produced by thermophilic anaerobic bacteria of No.
Glucoamylase can be produced from a thermophilic anaerobic bacterium belonging to the genus Clostridium, No. 8737. In order to improve the drawbacks of the conventional saccharification methods described above, the present inventors searched for enzyme-producing microorganisms with the aim of obtaining a new glucoamylase that has high heat resistance under acidic conditions. As a result, a moderately thermophilic anaerobic bacterium G-00003 was isolated from organic waste mesophilic methane fermentation slurry.
We have discovered that this method produces a new thermostable glucoamylase that satisfies the above conditions. Then, the present enzyme was isolated from the culture solution of the present bacterium, and as a result of intensive studies on a method for saccharifying starch using this enzyme, the present invention was achieved. The first feature of the present invention is that the novel thermostable glucoamylase according to the present invention is used to saccharify the raw material starch liquid without adding a calcium agent and without neutralizing the raw material starch liquid while keeping it acidic. The second feature is that the liquefied liquid is liquefied using an acid-resistant, low-calcium-requiring α-amylase, and is saccharified using an acid-resistant, heat-resistant glucoamylase. Therefore, saccharification can be performed at high speed without substantially neutralizing, and the load on the desalting process after saccharification is greatly reduced, and the proliferation of bacteria in the sugar production line can also be prevented. The third feature of the present invention is that starch is liquefied and saccharified in the simultaneous presence of α-amylase, which has heat resistance, acid resistance, and low calcium requirement, and the glucoamylase according to the present invention. The type of starch that can be used as a starting material for glucose in the present invention is not particularly limited.
For example, it is fully applicable to known starches such as potato, sweet potato, corn, tapioki, and wheat. The starch liquefaction method may be an enzymatic method using α-amylase or a chemical acid hydrolysis method. Addition of a calcium agent for the purpose of maintaining the heat resistance of the α-amylase according to the present invention is not necessary unless distilled water is used as the charging water and reagent grade amylose that has been completely decalcified is used. Therefore, if ordinary tap water is used, there is virtually no need for a subsequent desalination step for the purpose of removing cations. PH adjustment during liquefaction is usually not necessary because it almost coincides with the optimal reaction range and stability range of this α-amylase. Therefore, acidic starch slurry can be reacted without adjusting the pH. The raw material starch concentration varies depending on the type and quality of the raw material, and the use of the liquefaction treatment liquid, but it is 10 to 40% of the conventionally known concentration.
is within the range of The reaction temperature is determined based on the heat resistance of this α-amylase.
Used at temperatures below 100℃. Considering the reaction rate and heat resistance of the enzyme, it is preferable to carry out the reaction at 70 to 80°C.
The amount of enzyme added varies depending on the type of raw material starch, residence time, reaction temperature, pH, etc. in the reaction tank, but when the treatment time is fixed at 0.5 hours, it is 80℃,
To hydrolyze potato starch to the limit dextrin at pH 4.5, approximately 10 5 units per kg of starch are required. Here, the unit of α-amylase activity is
This is based on the blue value method, which will be explained in detail in the section on enzyme properties. The liquefied starch product, ie, dextran, that can be used in the present invention may be liquefied by an enzymatic method or by a chemical partial acid decomposition treatment. Adjustment of pH during saccharification is carried out during liquefaction treatment.
It is usually not necessary as long as it is carried out at an acidity of 4 to 5.
The concentration of the liquefaction treatment liquid varies depending on the type, quality, and purpose of the raw material, but is within the conventionally known range of 10 to 40%. The reaction temperature is 100° C. or lower due to the heat resistance of the glucoamylase according to the present invention. Considering the reaction rate and heat resistance of the enzyme, it is preferable to carry out the reaction at 60 to 80°C. Addition of calcium ions is unnecessary due to the characteristics of this enzyme. The amount of enzyme added varies depending on the quality and concentration of the raw material liquefaction treatment liquid in the reaction tank, residence time, reaction temperature, pH, etc., but the maximum dextrin saccharification rate is 90% at a treatment time of 70℃ and PH4.5. Approximately 120 units are required per 1 kg of potato starch in the pharmacopoeia for saccharification. Here, the definition of the activity unit of glucoamylase will be explained in detail in the section on the characteristics of the enzyme. When performing liquefaction and saccharification in the simultaneous presence of this α-amylase and this glucoamylase, the temperature, pH, etc. are within the same range for both enzymes, so conditions should be selected appropriately taking into account the characteristics of both enzymes. Do it below. Bacteria belonging to the genus Clostridium (Clostridium sp.
RS-0001) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (accession number: FERM P-7918). First, the details of the mycological properties of this bacterium will be explained. A. Morphological properties (1) Morphology of vegetative cells When cultured on an agar plate in the following starch/peptone medium in an anaerobic atmosphere at 60°C for 2 days, vegetative cells have a size of 0.4 to 0.8 x 2 to 5 μm. It is a straight-shaped bacillus. When cultured for 3 days or more, vegetative cells with the above-mentioned shape exist singly, and vegetative cells in chains also occur. The same holds true for liquid culture. Composition of starch/peptone medium Soluble starch 1.5% Peptone 0.5% Yeast extract 0.5% KH 2 PO 4 0.7% Na 2 HPO 4 0.35% MgSO 4.7H 2 O 0.001% Agar 2.0% Sodium thioglycolate 0.1% Water supply Water PH6.4 (2) Presence of spores Spore formation is observed in agar plate culture and liquid culture in starch/peptone medium. B. Culture characteristics (1) Colony morphology Colonies grown on agar plate in starch/peptone medium are flat and circular with a slightly raised center.
The periphery is the entire edge. No pigment formation is observed, and the surface is milky and opaque with gloss. It also has adhesive properties. (2) Agar plate culture and puncture culture on meat juice medium produce colonies similar to those on starch/peptone medium. Meat juice agar medium composition Meat extract 1.0% Peptone 1.0% Salt 0.2% Sodium thioglycolate 0.1% Agar 1.5% Distilled water PH6.0 (3) Puncture culture in meat juice medium Growth occurs with the generation of gases including hydrogen and carbon dioxide However, for this reason, the agar medium is divided into two or three places. (4) Meat juice liquid culture Grows only under anaerobic air, and the culture liquid becomes cloudy. Composition of meat juice medium Meat extract 1.0% Sodium thioglycolate 0.1% Distilled water PH6.0 (5) Meat juice/gelatin culture No growth observed. Composition of meat juice/gelatin medium Meat extract 1.0% Peptone 1.0% Salt 0.2% Gelatin 15% Sodium thioglycolate 0.1% Distilled water PH6.0 (3) Litmus milk culture Coagulates solidly with gas generation, due to acid production Turns red. C Physiological properties (1) Growth temperature range: Grows at 40-63℃. No growth was observed at 30℃, and growth was good at around 60℃. (2) PH range for growth: PH5-7. Around 5.6 is good. (3) Attitude towards oxygen Obligate anaerobic (4) O-F test (Hugh Laifson modified method) No growth was observed in the air, so the test was negative. Bacteria grow under anaerobic conditions with a layer of liquid paraffin,
Acid is produced and the culture solution turns yellow. Medium composition Peptone 0.2% Glucose 1.0% Salt 0.5% K 2 HPO 4 0.03% Sodium thioglycolate 0.1% Bromucresol purple 0.002% Agar 0.3% Distilled water PH6.0 Negative. (5) VP test negative. (6) MR test Anode changes to red. (7) Indole production Cannot be measured because it does not grow in peptone water. (8) Hydrogen sulfide formation Negative (when using Kligrer's medium). (9) Hydrolysis of starch Positive. It breaks down not only soluble starches but also granular starches such as potato starch. (10) Utilization of citric acid Negative (when using Simmons medium). (11) Use of ammonium salts Cannot be measured because they do not grow in peptone water. (12) Extracellular production of pigment Negative. (13) Urease negative. (14) Oxidase activity negative. (15) Catalase activity negative. (16) Sugar assimilation The table below shows the observation results of sugar assimilation and the presence or absence of gas generation using a Durham tube.
【表】
(17) 無機塩培地への生育
生育認められず。
(18) 有機酸の生成
各種培地から生成する有機酸組成を第２表に示
す。[Table] (17) Growth on mineral salt medium No growth observed. (18) Production of organic acids Table 2 shows the composition of organic acids produced from various media.
【表】【table】
【表】
供試液体培地の組成
炭素源 1.0％
ペプトン 1.0％
食 塩 0.2％
チオグリコール酸ナトリウム 0.1％
蒸留水
PH6.4
これらの結果をHoldemanの嫌気性細菌分類マ
ニユアルに照合したところ、嫌気性であり、桿菌
であり且つ胞子を形成することからクロスツリジ
ウム属に属する細菌と同定されたが、生育温度範
囲と糖の資化性及びガスの生成状況を全て具備す
るものが公知のどの菌種にもなく、新種の細菌と
判定した。
次に、本発明なる耐熱性α−アミラーゼの酵素
的特性について記す。
尚、α−アミラーゼ活性の測定方法は次のよう
に行つた。
Blue value法（日本化学会編：実験化学講座
24巻、生物化学、p279、丸善書店、1969）に
よる糊精化力を測定した。本法は、殿粉の分子が
加水分解されるのに伴い、殿粉−より素complex
に基づく青色の発色量が、分子量の低下に比例し
て減少する原理を応用したものである。まず、２
mg／mlの殿粉溶液２ml及び0.1Mくえん酸緩衝液
（PH4.0）１mlを試験管に取り、60℃水浴中で５分
間振盪した。次いで、粗酸素液として培養濾液１
mlを加え、30分間反応させた。反応後、反応液
0.4mlを採取し、直ちに0.5M酢酸溶液２mlと混合
して酵素反応を停止させた。次のその１mlを10ml
の1/3000Ｎよう素溶液中に加え、680nmでの吸光
度を分光光度計を用いて測定した。一方、酵素液
を加えた直後の反応液を採取して同様に発色さ
せ、吸光度を測定した。なお、殿粉としては重合
度約2000のアミロースを用いた。
α−アミラーゼ活性は次式により算出した。
α−アミラーゼ活性（単位）＝0time反応液の吸光度−3
0分反応液の吸光度／0time反応液の吸光度×10
(1) 作用及び基質特異性
本酵素は、馬鈴しよ，とうもろこし，甘しよ等
の殿粉を加水分解する液体型α−アミラーゼであ
る。
(2) 至適PH
第１図に、従来公知の代表的なα−アミラーゼ
の作用PH曲線を示す。曲線４で示した小笠原等の
バチルス・ズブチリス（J.Biochem.67．65.1970
年）及び曲線６で示した斉藤等のバチルス・リチ
エニホルミス（特開昭48−35083号公報）を起源
とするα−アミラーゼは、PH４〜11に好適域を有
する（最適PHでの活性の80％を有するPH域とす
る）。従来公知の酸性α−アミラーゼのうち、最
も酸性側で活性の高い田中等によるバチルス・リ
チエニホルミス起源α−アミラーゼ（特開昭52−
151970号公報、曲線３）では、好適域が3.5〜6.3
にあり、PH２で全く活性を示さない。
これに対し、本発明なるα−アミラーゼ（曲
線１）ならびにα−アミラーゼ（曲線２）の60
℃における最適PH域は、いずれも４付近にあり、
かつ好適PHはそれぞれ２〜5.7，２〜6.3にあつ
て、従来の酸性α−アミラーゼにくらべ、さらに
酸性側でも高い活性を有する。すなわち、PH２で
は、従来の酸性α−アミラーゼが全く活性を示さ
ないのに対し、本発明のα−アミラーゼはそれぞ
れ95％，81％の高い活性を示す。
なお、酵素反応は次の反応系を用いた。
酵素液：0.6〜1.3μｍ／ml
基質：アミロース１mg／ml
クエン酸緩衝液：0.025M
上述したように、本発明α−アミラーゼは従来
の酸性α−アミラーゼと作用PH域を異にすること
から、新しいα−アミラーゼであることは明らか
である。
(3) PH安定性
本発明で用いるα−アミラーゼ及びを、PH
２，４，６，７の各PH（0.025Mクエン酸緩衝液）
下で、60℃，30分間インキユベートした。反応液
を稀釈してPHを4.0に調整し、アミロースを基質
として残存活性を測定した。その結果両α−アミ
ラーゼは、上記のPH処理で完全に活性が保持され
ていた。したがつて、本α−アミラーゼは酸性域
でも安定性が高い特徴を有している。
(4) 至適温度
第２図に示す如く、本発明α−アミラーゼ
（曲線１１）及び（曲線１２）の至適PH4.0にお
ける至適温度は、いずれも80℃付近である。好適
温度（最適温度での活性の80％を有する温度域と
する）は65〜87％である。なお、反応にはくえん
酸緩衝液0.025Mを用いた。
(5) 熱安定性
本発明で用いるα−アミラーゼをPH6.0で
20μM塩化カルシウムの存在下に60〜97℃に加熱
処理し、残存活性を測定した。これをもとに各温
度における活性半減期を求め、その結果を第３図
に示す。80℃及び90℃における活性半減期（基質
無添加）はそれぞれ８時間，0.5時間であり、熱
安定性にすぐれている。α−アミラーゼについ
ても90℃における活性半減期は約0.5時間と、α
−アミラーゼと同等の耐熱性を有する。一方、
従来のα−アミラーゼの例とし、バチルス・リチ
エニホルミスに属するα−アミラーゼ生産菌、及
びバチルス・ズブチリスに属するα−アミラーゼ
生産菌の培養液から調製した部分精製α−アミラ
ーゼ標品を用い、カルシウム濃度20mMにおいて
半減期を実測した。その結果を第３図に付記す
る。反応は、くえん酸緩衝液を用いて、両α−ア
ミラーゼの最適PHである6.0で行つた。前者の80
℃における半減期は0.6時である。本発明で用い
るα−アミラーゼの耐熱性（曲線２１）は、従来
公知のサーマス属の耐熱性α−アミラーゼには及
ばないが、バチルス属のα−アミラーゼ（バチル
ス・リチエニホルシスSP.を起源とする耐熱性α
−アミラーゼ，曲線２２）とくらべて遜色ない。
(6) 液熱性に及ぼす金属塩の影響
本発明で用いるα−アミラーゼの耐熱性に及
ぼす金属塩の影響を第３表に示す。α−アミラー
ゼの水溶液に各種の金属塩を5mM濃度になる
様に添加し、加熱処理を行つて活性を測定した。
そして、加熱処理前に対する加熱処理後の活性、
すなわち残存活性を％で表示した。加熱処理及び
活性測定は以下の条件で行つた。
加熱処理条件
PH6.0
加熱温度：80％
保持時間：30分[Table] Composition of test liquid medium Carbon source 1.0% Peptone 1.0% Salt 0.2% Sodium thioglycolate 0.1% Distilled water PH6.4 When these results were compared with Holdeman's Anaerobic Bacteria Classification Manual, it was found to be anaerobic. Since it is a bacillus and forms spores, it was identified as a bacterium belonging to the genus Clostridia, but which bacterial species are known to have all the characteristics of the growth temperature range, sugar assimilation ability, and gas production. It was quickly determined that it was a new type of bacteria. Next, the enzymatic properties of the thermostable α-amylase of the present invention will be described. The α-amylase activity was measured as follows. Blue value method (edited by the Chemical Society of Japan: Experimental Chemistry Course)
24, Biochemistry, p279, Maruzen Shoten, 1969). In this method, as starch molecules are hydrolyzed, starch becomes more complex.
This is based on the principle that the amount of blue color produced decreases in proportion to the decrease in molecular weight. First, 2
2 ml of mg/ml starch solution and 1 ml of 0.1 M citrate buffer (PH4.0) were placed in a test tube and shaken in a 60°C water bath for 5 minutes. Next, culture filtrate 1 was used as a crude oxygen solution.
ml was added and allowed to react for 30 minutes. After reaction, reaction solution
0.4 ml was taken and immediately mixed with 2 ml of 0.5M acetic acid solution to stop the enzyme reaction. 10ml of the next 1ml
was added to a 1/3000N iodine solution, and the absorbance at 680 nm was measured using a spectrophotometer. On the other hand, the reaction solution immediately after adding the enzyme solution was sampled, developed in the same manner, and the absorbance was measured. Note that amylose with a degree of polymerization of about 2000 was used as the starch. α-amylase activity was calculated using the following formula. α-amylase activity (unit) = absorbance of 0time reaction solution - 3
Absorbance of 0-minute reaction solution / Absorbance of 0-time reaction solution x 10 (1) Action and substrate specificity This enzyme is a liquid α-amylase that hydrolyzes starches of potato, corn, sweet potato, etc. be. (2) Optimal PH Figure 1 shows the action PH curve of a typical α-amylase known in the art. Bacillus subtilis of Ogasawara et al. (J.Biochem. 67.65.1970 ) shown by curve 4
α-amylase originating from Bacillus lichieniformis (Japanese Unexamined Patent Publication No. 48-35083) by Saito et al., shown in Curve 6), has a suitable range between PH4 and 11 (80% of its activity at the optimum pH). ). Among the conventionally known acidic α-amylases, α-amylase derived from Bacillus lithieniformis (Japanese Unexamined Patent Application Publication No. 1983-1999) was developed by Tanaka, which is the most active on the acidic side.
151970, curve 3), the preferred range is 3.5 to 6.3.
It shows no activity at PH2. In contrast, α-amylase (curve 1) and α-amylase (curve 2) according to the present invention
The optimal pH range at ℃ is around 4,
Moreover, the preferable pH is 2 to 5.7 and 2 to 6.3, respectively, and the enzyme has higher activity even on the acidic side than conventional acidic α-amylase. That is, at PH2, conventional acidic α-amylase shows no activity at all, whereas the α-amylase of the present invention shows high activity of 95% and 81%, respectively. In addition, the following reaction system was used for the enzyme reaction. Enzyme solution: 0.6-1.3μm/ml Substrate: amylose 1mg/ml Citrate buffer: 0.025M As mentioned above, the α-amylase of the present invention has a different action PH range from that of conventional acidic α-amylase. It is clear that this is a new α-amylase. (3) PH stability α-amylase used in the present invention and PH stability
PH of 2, 4, 6, 7 (0.025M citrate buffer)
The cells were incubated at 60°C for 30 minutes. The reaction solution was diluted to adjust the pH to 4.0, and the residual activity was measured using amylose as a substrate. As a result, the activity of both α-amylases was completely retained by the above PH treatment. Therefore, the present α-amylase is characterized by high stability even in an acidic region. (4) Optimal temperature As shown in FIG. 2, the optimal temperatures of the α-amylases of the present invention (curve 11) and (curve 12) at the optimal pH of 4.0 are both around 80°C. The preferred temperature (temperature range having 80% of the activity at the optimum temperature) is 65-87%. Note that 0.025M citric acid buffer was used in the reaction. (5) Thermostability α-amylase used in the present invention at pH 6.0
Heat treatment was performed at 60-97°C in the presence of 20 μM calcium chloride, and residual activity was measured. Based on this, the activity half-life at each temperature was determined, and the results are shown in FIG. The half-life of activity at 80°C and 90°C (without substrate added) is 8 hours and 0.5 hours, respectively, indicating excellent thermal stability. The activity half-life of α-amylase at 90°C is approximately 0.5 hours,
- Has heat resistance equivalent to amylase. on the other hand,
As an example of conventional α-amylase, a partially purified α-amylase preparation prepared from a culture solution of α-amylase-producing bacteria belonging to Bacillus lithieniformis and α-amylase producing bacteria belonging to Bacillus subtilis was used, and the calcium concentration was 20mM. The half-life was actually measured. The results are added to Figure 3. The reaction was carried out using citrate buffer at 6.0, which is the optimum pH for both α-amylases. 80 of the former
The half-life at °C is 0.6 hours. The heat resistance of the α-amylase used in the present invention (curve 21) is not as good as that of the conventionally known thermostable α-amylase of the genus Thermus, but it is Sex α
- Comparable to amylase, curve 22). (6) Effect of metal salts on heat resistance of liquid The effect of metal salts on the heat resistance of α-amylase used in the present invention is shown in Table 3. Various metal salts were added to an aqueous solution of α-amylase at a concentration of 5mM, heat-treated, and the activity was measured.
And the activity after heat treatment compared to before heat treatment,
That is, the residual activity was expressed in %. Heat treatment and activity measurement were performed under the following conditions. Heat treatment conditions PH6.0 Heating temperature: 80% Holding time: 30 minutes
【表】
活性測定は、試料液を希釈後、以下の条件下で
行つた。なお、各金属塩を本添加濃度で添加して
も、活性測定に影響のないことを確認している。
活性測定条件
PH4.0（0.025Mくえん酸緩衝液）
活性測定温度：60℃
第１表から明らかに、カルシウムイオンに保護
効果が認められるのに対し、ナトリウム，カケ
ル，コバルト，亜鉛及びマンガンの各イオンは耐
熱性を低下させる。また、本α−アミラーゼは
0.5μMのEDTAで耐熱性を失うことも確認してい
る。
本α−アミラーゼのカルシウム要求濃度は第４
図の曲線３１に示すように、100μM（4ppm）で
あり、水道水中のカルシウム濃度で十分安定化さ
れる。さらに本酵素は1μM以下のカルシウム濃
度においても65％の活性を保持している。また、
α−アミラーゼもα−アミラーゼと同等のカ
ルシウム要求性を有している。
これに対し、バチルス・リチエニホルミスに属
するα−アミラーゼ生産菌から部分精製したα−
アミラーゼは、第４図の曲線３２に示すように、
30mMのカルシウムイオンを必要とする。なお、
加熱処理は両酵素ともPH６，80℃で30分間加熱
し、活性測定は各々の最適にて60℃で行つた。
一方、バチルス・ズブチリスの耐熱性α−アミ
ラーゼでは、カルシウム必要濃度は３〜10mMで
ある（特開昭51−44690号公報、特開昭58−34117
号公報）。
したがつて、本発明α−アミラーゼは、従来公
知の耐熱性α−アミラーゼに比べ著しくカルシウ
ム要求性が低い。
(7) 精製方法
実施例において詳述するので、ここでは簡単な
説明にとどめる。
本発明で用いるα−アミラーゼ生産菌を、殿
粉，ペプトン及び酵母エキスを含有する液体培地
に接種し、嫌気条件下で60℃に１〜３日間培養す
る。培養液を遠心分離等により菌体及びそれ以外
の不溶物資を除したいわゆる培養濾液を得る。次
いで、培養濾液を、モレキユラシーブ膜濾過，イ
オン交換クロマト，ゲル濾過クロマト，塩析等の
公知の方法を適宜利用して、本発明α−アミラー
ゼを濃縮するとともにそれ以外の不純物を除く。
(8) 分子量
本発明α−アミラーゼの分子量は未確認である
が、モレキユラシーブ膜濾過における挙動から、
分子量は20000以上と推定される。
以上述べたことから明らかなように本発明で用
いる耐熱性α−アミラーゼは、特に作用PH並びに
カルシウム要求性において、従来の嫌気性細菌の
生産する耐熱性酵素と著しく異なる。
次に、本発明で用いるグルコアミラーゼを産生
するClostridium spG−0005は工業技術院微生物
工業技術研究所に寄託している（受託番号：；微
工研菌寄第8737号（FERM Ｐ−8737））。まず、
本菌の菌学的性質の詳細を説明する。なお、以下
の記載において％は特に指示しない限り重量％で
ある。
Ａ 形態的性質
(1) 栄養細胞の形態
下記のデキストリン・ペプトン培地の寒天平板
上、嫌気性範囲気中、60℃で２日間培養した場
合、栄養細胞は0.3〜0.5×２〜4μmの大きさの直
状の桿菌である。上記の栄養細胞が単独あるいは
連鎖して存在する。液体培養においても同様とな
る。
デキストリン・ペプトン培地の組成
デキストリン 1.5％
ペプトン 0.5％
酵母エキス 0.5％
KH2PO4 0.7％
Na2HPO4 0.2％
MgSO4・7H2Ｏ 0.001％
寒 天 2.0％
オチグリコール酸ナトリウム 0.1％
水道水
PH6.0
(2) 運動性の有無
運動性は認められない
(3) 胞子の有無
殿粉・ペプトン培地の寒天平板培養において胞
子の形成が認められる。胞子は栄養細胞内端部に
形成され、直径0.5μｍ程度の球状をなしている。
(4) グラム染色性
陰性
Ｂ 培養的特性
(1) コロニーの形態
殿粉・ペプトン培地の寒天平板培養でのコロニ
ーは、中心部がやや隆起した扁平な円形となり、
周縁部は不規則である。色素は産生せず、乳白色
半透明である。
(2) 肉汁寒天平板培養
生育は認められない
肉汁寒天培地組成
肉エキス 1.0％
ペプトン 1.0％
食 塩 0.2％
チオグリコール酸ナトリウム 0.1％
寒 天 2.0％
蒸留水
PH6.0
(3) 肉汁寒天斜面培養
生育は認められない
(4) 肉汁寒天穿刺培養
穿刺線にそつてわずかに増殖していることが観
察される。色素の産生は認められない。
(5) 肉汁液体培養
生育は認められない
肉汁培地の組成
肉エキス 1.0％
ペプトン 1.0％
食 塩 0.2％
チオグリコール酸ナトリウム 0.01％
蒸留水
PH6.0
(6) 肉汁・ゼラチン培養
生育は認められない。ゼラチンの液化も認めら
れない。
肉汁・ゼラチン培地の組成
肉エキス 1.0％
ペプトン 1.0％
食 塩 0.2％
ゼラチン 15.0％
チオグリコール酸ナトリウム 0.1％
蒸留水
PH6.0
(7) リトマスミルク培養
酸を生成して赤変し、固く凝固する。ガスの発
生が認められる。
Ｃ 生理的性質
(1) 生育の温度範囲
49〜64℃で生育する。46℃，69℃では生育は認
められない。57〜61℃付近で良好に生育する。
(2) 生育のPH範囲
PH4.8〜7.5で生育する。PH6.0付近で良好に生育
する。
(3) 酸素に対する態度
偏性嫌気性
(4) Ｏ−Ｆテスト（Hugh Laifson法）
陰性。空気雰囲気中及び流動パラフイン重層に
よる嫌気性条件下共にガス生成を伴つて生育し、
酸の生成により黄色となる。空気雰囲気中での培
養においては、気相境界部より約１cm下より底部
にかけて生育した。
培地組成
ペプトン 0.2％
ぶどう糖 1.0％
食 塩 0.5％
K2HPO4 0.03％
ブロムクレゾールパープル 0.002％
寒 天 0.3％
蒸留水
PH6.0
(5) 硝酸塩の還元
陰性
(6) VPテスト
陰性
(7) MRテスト
陽性。赤変化する。
(8) インドール生成
ペプトン水に生育しないため測定できない。
(9) 硫化水素の生成
陰性（Kligrerの培地使用において）。
(10) 殿粉の加水分解
陽性。
(11) クエン酸の利用
陽性（Simmons培地使用において）。
(12) アンモニウム塩の利用
ペプトン水に生育せず、測定できない。
(13) 色素の菌体外生成
陰性。
(14) ウレアーゼ
陰性。
(15) オキシダーゼ
陰性。
(16) カタラーゼ活性
陰性。
(17) 糖の資化性
糖の資化性及びダーラム管を用いたガス発生有
無の観察結果を第１表に示す。表中、資化性及び
ガスの生成が認められた場合には＋，認められな
い場合には−の記号で示した。[Table] Activity measurements were performed under the following conditions after diluting the sample solution. It has been confirmed that the addition of each metal salt at this concentration does not affect the activity measurement. Activity measurement conditions: PH4.0 (0.025M citrate buffer) Activity measurement temperature: 60℃ From Table 1, it is clear that calcium ions have a protective effect, while sodium, cobalt, zinc, and manganese each have a protective effect. Ions reduce heat resistance. In addition, this α-amylase is
It has also been confirmed that heat resistance is lost at 0.5 μM EDTA. The calcium requirement concentration of this α-amylase is the fourth
As shown by curve 31 in the figure, it is 100 μM (4 ppm) and is sufficiently stabilized by the calcium concentration in tap water. Furthermore, this enzyme retains 65% activity even at calcium concentrations of 1 μM or less. Also,
α-amylase also has the same calcium requirement as α-amylase. In contrast, partially purified α-amylase-producing bacteria belonging to Bacillus lithieniformis
As shown in curve 32 of FIG. 4, amylase is
Requires 30mM calcium ions. In addition,
Both enzymes were heated at pH 6 and 80°C for 30 minutes, and activity measurements were carried out at 60°C at the optimum temperature for each enzyme. On the other hand, in the thermostable α-amylase of Bacillus subtilis, the required concentration of calcium is 3 to 10 mM (Japanese Patent Laid-Open No. 51-44690, Japanese Patent Laid-Open No. 58-34117).
Publication No.). Therefore, the α-amylase of the present invention has a significantly lower calcium requirement than conventionally known heat-stable α-amylases. (7) Purification method This will be explained in detail in Examples, so a brief explanation will be given here. The α-amylase-producing bacteria used in the present invention are inoculated into a liquid medium containing starch, peptone, and yeast extract, and cultured at 60° C. for 1 to 3 days under anaerobic conditions. A so-called culture filtrate is obtained by removing bacterial cells and other insoluble materials from the culture solution by centrifugation or the like. Next, the culture filtrate is appropriately used to concentrate the α-amylase of the present invention and to remove other impurities using known methods such as molecular sieve membrane filtration, ion exchange chromatography, gel filtration chromatography, and salting out. (8) Molecular weight Although the molecular weight of the α-amylase of the present invention has not been confirmed, based on its behavior in molecular sieve membrane filtration,
The molecular weight is estimated to be over 20,000. As is clear from the above, the thermostable α-amylase used in the present invention is significantly different from conventional thermostable enzymes produced by anaerobic bacteria, particularly in terms of action pH and calcium requirement. Next, Clostridium spG-0005, which produces the glucoamylase used in the present invention, has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Accession number: FERM P-8737). . first,
The details of the mycological properties of this bacterium are explained below. In the following description, % is by weight unless otherwise specified. A Morphological properties (1) Morphology of vegetative cells When cultured on an agar plate in the following dextrin-peptone medium in an anaerobic atmosphere at 60°C for 2 days, the vegetative cells have a size of 0.3 to 0.5 x 2 to 4 μm. It is a straight bacillus. The above vegetative cells exist singly or in a chain. The same applies to liquid culture. Composition of dextrin /peptone medium Dextrin 1.5% Peptone 0.5% Yeast extract 0.5% KH 2 PO 4 0.7% Na 2 HPO 4 0.2% MgSO 4.7H 2 O 0.001% Agar 2.0% Sodium othiglycolate 0.1% Tap water PH6. 0 (2) Presence or absence of motility No motility observed (3) Presence or absence of spores Spore formation is observed in agar plate culture of starch/peptone medium. Spores are formed at the inner end of vegetative cells and are spherical with a diameter of about 0.5 μm. (4) Gram staining negative B Culture characteristics (1) Colony morphology Colonies cultured on agar plate in starch/peptone medium are flat circular with a slightly raised center.
The periphery is irregular. It does not produce any pigment and is milky white and translucent. (2) Meat juice agar plate culture No growth observed Meat juice agar medium composition Meat extract 1.0% Peptone 1.0% Salt 0.2% Sodium thioglycolate 0.1% Agar 2.0% Distilled water PH6.0 (3) Meat juice agar slant culture Growth (4) Meat juice agar puncture culture Slight growth is observed along the puncture line. No pigment production is observed. (5) Meat juice liquid culture No growth observed Meat juice culture medium composition Meat extract 1.0% Peptone 1.0% Salt 0.2% Sodium thioglycolate 0.01% Distilled water PH6.0 (6) Meat juice/gelatin culture No growth observed. No liquefaction of gelatin was observed. Composition of meat juice/gelatin medium Meat extract 1.0% Peptone 1.0% Salt 0.2% Gelatin 15.0% Sodium thioglycolate 0.1% Distilled water PH6.0 (7) Litmus milk culture Produces acid, turns red, and solidifies. Gas generation is observed. C Physiological properties (1) Growth temperature range: Grows at 49-64℃. No growth is observed at 46°C or 69°C. Grows well around 57-61℃. (2) Growth PH range: Grows at PH4.8 to 7.5. Grows well around PH6.0. (3) Attitude towards oxygen Obligate anaerobic (4) O-F test (Hugh Laifson method) Negative. Grows with gas production both in air atmosphere and under anaerobic conditions with a layer of liquid paraffin,
It turns yellow due to the formation of acid. When cultured in an air atmosphere, growth occurred from about 1 cm below the gas phase boundary to the bottom. Medium composition Peptone 0.2% Glucose 1.0% Salt 0.5% K 2 HPO 4 0.03% Bromcresol purple 0.002% Agar 0.3% Distilled water PH6.0 (5) Nitrate reduction Negative (6) VP test Negative (7) MR test Positive. Changes to red. (8) Indole production Cannot be measured because it does not grow in peptone water. (9) Hydrogen sulfide formation Negative (when using Kligrer's medium). (10) Hydrolysis of starch Positive. (11) Utilization of citric acid Positive (when using Simmons medium). (12) Use of ammonium salts It does not grow in peptone water and cannot be measured. (13) Extracellular production of pigment Negative. (14) Urease negative. (15) Oxidase negative. (16) Catalase activity negative. (17) Sugar assimilation ability Table 1 shows the observation results of sugar assimilation ability and the presence or absence of gas generation using a Durham tube. In the table, when assimilation and gas production were observed, they were indicated by a + symbol, and when they were not observed, they were indicated by a - symbol.
【表】【table】
【表】
糖資化性試験用液体培地組成
炭素源（糖） 1.0％
ペプトン 1.0％
NaCl 0.2％
チオグリコール酸ナトリウム 0.1％
蒸留水
PH6.0
(18) 無機塩培地への生育
生育は認められない。
これらの結果をバージーの細菌分類マニユアル
（Bergey′s Manual of Determinative
Bacteriology 8th Edition）に照合したところ、
桿菌であり、嫌気性であり且つ硫化水素の生成が
陰性であることからクロスツリジウム属に属する
細菌と同定されたが、生育温度範囲と糖の資化性
とを全て具備するものが公知のどの菌種にもな
く、新種の細菌と判定した。
次に、本発明なる耐熱性グルコアミラーゼの酵
素的特性について記す。
なお、グルコアミラーゼ活性の測定は次のよう
に行つた。
グルコアミラーゼ活性測定の基質には可溶性殿
粉（和光純薬製、生化学用）を用いた。まず、５
％可溶性殿粉溶液0.5ml，PH4.5の0.1M酢酸−酢酸
ナトリウム緩衝液0.5ml，純水１ml，酵素溶液0.5
mlを混合し、60℃で30分間酵素反応を行わせた。
次いで、反応液中のぶどう糖量をグルコース分析
器（米国，イエロー・スプリングス・インスツル
メント・カンパニー（Yellow Springs
Instrument Company）社製、グルコースオキシ
ダーゼ法により測定する。）を用いて測定した。
グルコアミラーゼ活性の１単位は、上記測定条件
下で１分間に1μmolのぶどう母を生成する能力と
定義した。
(1) 調製方法
実施例において詳細に説明するので、ここでは
簡単な説明にとどまる。
本発明になるクロスツリジウム属細菌Ｇ−0005
菌を、デキストリン，ペプトン及び酵母エキスを
含有する液体培地に接種し、嫌気条件下、60℃に
て１〜３日間培養する。培養液を遠心分離等によ
り菌体及びそれ以外の不溶物質を除去したいわゆ
る培養濾液を得る。次いで、培養濾液を、モレキ
ユラーシーブ膜濾過、塩析イオン交換クロマト、
ゲル濾過クロマト等、公知の方法を適宜利用して
本発明のグルコアミラーゼを濃縮するとともに、
それ以外の不純物を除く。
なお、液体培養における液体培地の炭素源とし
ては、上記デキストリンに限るものではなく、可
溶性殿粉，馬鈴薯，殿粉，コーンスターチ，甘し
よでん粉廃糖みつ等を用いてもよい。また、その
他の栄養成分も上記に限定するものではなく、コ
ーンステイープリカー，各種アミノ酸，ビタミ
ン，各種塩等を単独もしくは混合して用いてもよ
い。
(2) 作用及び基質特異性
本酵素は、馬鈴薯，とうもろこし，甘しよ等の
殿粉、およびこれを加水分解して得た可溶性殿
粉，デキストリン，マルトース等をぶどう糖に加
水分解するグルコアミラーゼである。マルトース
基質の場合、ぶどう糖生成速度は可溶性殿粉基質
の約1/2である。
(3) 至適PH
本酵素の60℃における作用PH曲線を第１図に示
す。本酵素の60℃における最適PH域は４〜５にあ
り、かつ好適PHは（最適PHでの活性の80％を有す
るPH域とする）3.8〜5.7にある。なお、反応の際
のPH緩衝液としては、塩化カリウム−塩酸（PH
2.0），グリシン−塩酸（PH2.5〜3.5），Ｐ：P′−ジ
メチルグルタル酸−トリスヒドロキシメチルアミ
ノメタン−２−アミノ−２−メチル−１：３−プ
ロパンジオール（以下「GTA」と略称）（PH3.5
〜９）の0.05M緩衝液を用いた。
(4) PH安定性
本発明のグルコアミラーゼをPH２，３，４，
4.5，５，６，７，９の各PH下（0.05酢酸−塩酸、
及び0.05M GTA緩衝液を使用）で70℃，30分間
インキユベートした。そののち、反応液を稀釈
し、PH4.5に調製したのち、可溶性殿粉を基質と
して残存活性を測定した。その結果を第２図に示
した。本発明のグルコアミラーゼはPH4.5〜5.0の
範囲で完全に活性が保持されていた。したがつ
て、本グルコアミラーゼは酸性領域ですぐれた安
定性を有する酵素であることがわかつた。
(5) 至適温度
本発明のグルコアミラーゼの活性をPH4.5の条
件下、40，50，60，65，70，75℃の温度にて測定
したところ、第３図に示す結果を得た。本結果よ
り、至適温度は70℃付近にある。好適温度（最適
温度での活性の80％を有する温度域とする）は53
〜73℃である。[Table] Composition of liquid medium for sugar assimilation test Carbon source (sugar) 1.0% Peptone 1.0% NaCl 0.2% Sodium thioglycolate 0.1% Distilled water PH6.0 (18) Growth on mineral salt medium No growth observed . These results were translated into Bergey's Manual of Determinative Bacteria.
Bacteriology 8th Edition).
It was identified as a bacterium belonging to the genus Clostridium because it is a bacillus, anaerobic, and negative for hydrogen sulfide production. It was determined to be a new type of bacteria, as it was not found in any other bacterial species. Next, the enzymatic properties of the thermostable glucoamylase of the present invention will be described. Note that glucoamylase activity was measured as follows. Soluble starch (manufactured by Wako Pure Chemical Industries, Ltd., for biochemical use) was used as the substrate for measuring glucoamylase activity. First, 5
0.5 ml of % soluble starch solution, 0.5 ml of 0.1 M acetic acid-sodium acetate buffer with pH 4.5, 1 ml of pure water, 0.5 ml of enzyme solution
ml was mixed and the enzymatic reaction was performed at 60°C for 30 minutes.
Next, the amount of glucose in the reaction solution was measured using a glucose analyzer (Yellow Springs Instrument Company, USA).
Measured by the glucose oxidase method manufactured by Instrument Company. ).
One unit of glucoamylase activity was defined as the ability to produce 1 μmol of grape mother per minute under the above measurement conditions. (1) Preparation method Since this will be explained in detail in Examples, only a brief explanation will be given here. Clostridium bacteria G-0005 according to the present invention
The bacteria are inoculated into a liquid medium containing dextrin, peptone, and yeast extract, and cultured at 60° C. for 1 to 3 days under anaerobic conditions. A so-called culture filtrate is obtained by removing bacterial cells and other insoluble substances from the culture solution by centrifugation or the like. Next, the culture filtrate was subjected to molecular sieve membrane filtration, salting out ion exchange chromatography,
While concentrating the glucoamylase of the present invention using appropriately known methods such as gel filtration chromatography,
Remove other impurities. In addition, the carbon source of the liquid medium in liquid culture is not limited to the above-mentioned dextrin, and soluble starch, potato, starch, corn starch, sweet starch molasses, etc. may be used. Further, other nutritional components are not limited to those mentioned above, and cornstarch liquor, various amino acids, vitamins, various salts, etc. may be used alone or in combination. (2) Action and substrate specificity This enzyme is a glucoamylase that hydrolyzes starches such as potato, corn, and sweet potato, as well as soluble starches obtained by hydrolyzing these, dextrin, maltose, etc., into glucose. be. For maltose substrates, the glucose production rate is approximately 1/2 that of soluble starch substrates. (3) Optimal PH Figure 1 shows the action PH curve of this enzyme at 60°C. The optimum pH range of this enzyme at 60°C is between 4 and 5, and the preferred pH range is between 3.8 and 5.7 (assuming the pH range has 80% of the activity at the optimum pH). The PH buffer used in the reaction is potassium chloride-hydrochloric acid (PH
2.0), glycine-hydrochloric acid (PH2.5-3.5), P:P'-dimethylglutaric acid-trishydroxymethylaminomethane-2-amino-2-methyl-1:3-propanediol (hereinafter abbreviated as "GTA") ) (PH3.5
~9) 0.05M buffer was used. (4) PH stability The glucoamylase of the present invention has a pH of 2, 3, 4,
Under each pH of 4.5, 5, 6, 7, 9 (0.05 acetic acid-hydrochloric acid,
and 0.05M GTA buffer) at 70°C for 30 minutes. Thereafter, the reaction solution was diluted and adjusted to pH 4.5, and residual activity was measured using soluble starch as a substrate. The results are shown in Figure 2. The glucoamylase of the present invention completely retained its activity within the pH range of 4.5 to 5.0. Therefore, the present glucoamylase was found to be an enzyme having excellent stability in an acidic region. (5) Optimal temperature The activity of the glucoamylase of the present invention was measured at temperatures of 40, 50, 60, 65, 70, and 75°C under the condition of PH4.5, and the results shown in Figure 3 were obtained. . From this result, the optimal temperature is around 70°C. The preferred temperature (the temperature range that has 80% of the activity at the optimal temperature) is 53
~73℃.
【表】
(6) グルコアミラーゼ活性に及ぼす金属塩の影響
本発明のグルコアミラーゼの活性に及ぼす金属
塩の影響を表４に示す。グルコアミラーゼ活性の
測定において各種の金属塩を5mMになるように
添加した。そして、金属塩無添加に対する活性を
％で表示した。
なお、アンモニウム塩及びEDTA添加の場合
についても表４中に附記した。マグネシウムイオ
ン，カルシウムイオン，カリウムイオンがグルコ
アミラーゼの活性化作用を有することが認められ
る。ニツケルイオン，鉄イオンには阻害作用が認
められる。[Table] (6) Effect of metal salts on glucoamylase activity Table 4 shows the effects of metal salts on the activity of glucoamylase of the present invention. In measuring glucoamylase activity, various metal salts were added to a concentration of 5mM. The activity was expressed in % relative to that without addition of metal salts. Note that the case of addition of ammonium salt and EDTA is also added in Table 4. Magnesium ions, calcium ions, and potassium ions are recognized to have an activating effect on glucoamylase. Nickel ions and iron ions have inhibitory effects.
【表】
活性測定条件
PH5.0（0.1Mクエン酸−第２クエン酸ナトリウ
ム緩衝液）活性測定温度：60℃
(7) 熱安定性
本発明のグリコアミラーゼを基質無添加下、PH
4.5にて、60〜80℃の加熱処理を行い、一定時間
毎（20，40秒，１，２，４，10，20，40分，１，
２，４，６，８，16時間，１，２，４，７，10，
15，20日）に処理液の一部を採取し、液中のグル
コアミラーゼ活性を60℃，PH4.5にて測定した。
これをもとに各温度における活性半減期を求め、
その結果を第４図に示した。70℃及び75℃におけ
る基質無添加下での活性半減期はそれぞれ、６時
間，１分間である。本グルコアミラーゼはこれま
で公知のグルコアミラーゼに較べ高度の耐熱性を
有している。一方、PH4.0，4.3，4.5，5.0，6.0の
各PHにおける基質無添加下、70℃での活性半減期
を求め、第３表に示した。この結果から、本グル
コアミラーゼは、クロスツリジウム・サーモアミ
ロリテイクム（Clostridium
thermoamylolyticum）、サーモマイセス・ラヌ
ギノスス（Thermomyces lanuginosus）、及び
タラロミセス・デユポンテイ（Talaromyces
duponti）により産生されるグルコアミラーゼよ
りも特に、PH４〜５の酸性領域においてすぐれた
耐熱性をもつことを示す。
(8) 耐熱性に及ぼす金属塩の影響
本発明のグルコアミラーゼの耐熱性に及ぼす金
属塩の影響を第５表に示す。グルコアミラーゼの
水溶液（0.05M 酢酸−酢酸ナトリウム緩衝液に
溶解、PH4.5）に各種の金属塩を5mM濃度になる
ように添加し、70℃，１時間の加熱処理を行つた
のち、PH4.5、60℃でクルコアミラーゼ活性を測
定した。そして、加熱処理前に対する加熱処理後
の活性、すなわち残存活性を％で表示した。
第５表から、ニツケルイオン，マンガンイオ
ン，マグネシウムイオンに保護効果のあることが
認められる。コバルトイオン，カルシウムイオン
については保護効果は認められない。亜鉛イオン
は耐熱性を著しく低下させる。[Table] Activity measurement conditions PH5.0 (0.1M citric acid-secondary sodium citrate buffer) Activity measurement temperature: 60℃ (7) Thermostability The glycoamylase of the present invention was measured at PH5.0 without the addition of a substrate.
In step 4.5, heat treatment was performed at 60 to 80℃, and the heat treatment was carried out at fixed intervals (20, 40 seconds, 1, 2, 4, 10, 20, 40 minutes, 1,
2, 4, 6, 8, 16 hours, 1, 2, 4, 7, 10,
A portion of the treated solution was collected on days 15 and 20), and the glucoamylase activity in the solution was measured at 60°C and PH4.5.
Based on this, determine the activity half-life at each temperature,
The results are shown in Figure 4. The half-lives of activity in the absence of substrate at 70°C and 75°C are 6 hours and 1 minute, respectively. This glucoamylase has a higher degree of thermostability than conventionally known glucoamylases. On the other hand, the half-lives of activity at 70°C without the addition of substrate at each pH of 4.0, 4.3, 4.5, 5.0, and 6.0 were determined and are shown in Table 3. Based on these results, this glucoamylase is suitable for Clostridium thermoamylolyteicum (Clostridium thermoamylolyteicum).
thermoamylolyticum), Thermomyces lanuginosus, and Talaromyces dupontei.
It has been shown to have superior heat resistance, especially in the acidic range of PH4 to 5, than glucoamylase produced by A. dupontii. (8) Effect of metal salts on heat resistance Table 5 shows the effects of metal salts on the heat resistance of the glucoamylase of the present invention. Various metal salts were added to an aqueous solution of glucoamylase (dissolved in 0.05M acetic acid-sodium acetate buffer, PH4.5) to a concentration of 5mM, and after heat treatment at 70°C for 1 hour, pH4. 5. Curcoamylase activity was measured at 60°C. The activity after the heat treatment, that is, the residual activity, was expressed in % compared to before the heat treatment. From Table 5, it is recognized that nickel ions, manganese ions, and magnesium ions have a protective effect. No protective effect was observed for cobalt ions and calcium ions. Zinc ions significantly reduce heat resistance.
ぶどう糖や異性化糖を製造するには、まず、原
料である殿粉をα−アミラーゼで液化し、その後
グルコアミラーゼで糖化するのであるが、液化の
際、原料の殿粉を20〜40％の高濃度に仕込むた
め、液のPHは酸性を呈する。このため、従来のα
−アミラーゼ及びグルコアミラーゼを用いる場合
には、作用PH域が中性域にあるため、アルカリで
中和しなければならない。これに対し本発明で用
いる耐熱耐酸性のα−アミラーゼ及びグルコアミ
ラーゼは、酸性域でも高活性を有することから、
液化での中和を行なうことなく酸性の状態のまま
（この場合PH調整も必らずしも必要とならない）
液化及び糖化を行なうことが可能であり、ひいて
は反応後の脱塩工程への負荷を大幅に軽減でき
る。
また、本発明で用いる新規なグルコアミラーゼ
は80℃以上の高温でも活性を有することから、80
℃の高温で液化した後、そのまま冷却しないで用
いることができる。
〔発明の実施例〕
以下、本発明の実施例を示し、さらに詳しく説
明する。
実施例 １
可溶性殿粉1.5％，ポリペプトン0.5％，酵母エ
キス0.5％，リン酸第１オリウム0.7％，リン酸第
２ソーダ0.35％、硫酸マグネシウム・ク水和物
0.01％，チオグリコール酸ナトリウム0.1％及び
水道水を含む液体培地（PH6.5）15.75Kgを、内容
積５の培養槽５基に3.15Kgずつ分注し、120℃
で20分間殺菌する。これに同上培地で嫌気的に培
養した本発明者等により分離せるクロスツリジウ
ム属の菌体懸濁液350ｇを各槽毎に添加した。次
いで、ガス出口に水封トラツプを付し、発酵槽内
気相部をアルゴンガスで十分置換後、遮気条件下
で培養する。培養液のPHは6.0に自動調整し、温
度も60℃に自動調整する。22時間培養後、培養物
を合せ6000rpmで遠心分離し、菌体を除去する。
この上澄液は60単位／ｂの比活性を示した。
次に、上記上澄液14.6Kgを２℃に冷却したの
ち、コーンスターチ300ｇ及びセライト（和光純
薬製）300ｇを入れ、攪拌下、10分間接触させた。
次いで、減圧濾過により、コーンスターチ及びセ
ライトを回収し、更に２℃に冷却した純水３で
洗浄した。次いで、あらかじめ、65℃にあたため
た5mMの塩化カルシウムを含む0.05M トリス、
塩酸緩衝液（PH7.5）に上記コーンスターチ及び
セライトを分散させ、水浴上で65℃に５分間保持
したのち、減圧濾過により固液分離し、更に固形
物を１の上記緩衝液（65℃）で洗浄し、濾過液
及び洗浄液を合わせて４を得た。次に上記濾過
を２℃に冷却後、モレキユラーシーブ膜（分画分
子量：20000）で濾過して濃縮し、更に濾過によ
り不溶物を除いて濃縮液400mlを得た。この濃縮
液は1.5×103単位／mlのα−アミラーゼ活性を示
した。
次に、上記濃縮液を、ジエチルアミノエチル化
架橋アガロースゲル（DEAEセフアロース・CL
−6B，フアルマツア社製）を用いたイオン交換
クロマト（カラムサイズ：φ50×210mm）により
精製した。まず、上記の濃縮液を、0.05Mトリス
塩酸緩衝液（PH7.5）で２回透析したのち、不溶
物を濾過し、同じ緩衝液で緩衝化したゲルカラム
にチヤージし、洗浄した。次いで、緩衝液中の塩
化ナトリウム濃度を直線勾配で上昇しつつ展開し
た。その結果、塩化ナトリウム濃度0.04Mと
0.08Mの位置にα−アミラーゼ活性を有する２つ
のピークが認められた。0.04Mの位置に溶出した
α−アミラーゼの活性量は吸着全活性の約30
％、0.08Mの位置に溶出したα−アミラーゼの
それは約60％であつた。両活性フラクシヨンを純
水中で透析後、凍結乾燥してα−アミラーゼ、
及びα−アミラーゼを得た。培養液の遠心上澄
基準の活性回収率は、それぞれ17％，32％であつ
た。
次にデキストリン1.5％，ポリペプトン0.5％，
酵母エキス0.5％，りん酸第１カリウム0.7％，り
ん酸第２ナトリウム0.2％，硫酸マグネシウム・
７水和物0.001％，チオグリコール酸ソーダ0.1％
及び水道水を含む液体培地（PH6.0）120Kgを内容
積20の培養槽10基に12Kgずつ分注し、120℃で
15分間殺菌した。それぞれの培養槽に本発明に係
るグルコアミラーゼ生産菌であるclostridium
SPG−0005（微工研菌寄第8737号）の菌体懸濁液
0.5Kgを添加した。次に、気相部分を窒素で置換
し、嫌気条件下、60℃，PH6.0で２日間培養した。
培養液を6000rpm，10分間遠心分離して菌体を除
去した。回収した上澄液をさらにガラス濾紙で濾
過し固形粒子フリーの培養濾液を得た。本液中に
は0.05単位／mlのグルコアミラーゼが含まれてい
る。
次に上記培養濾液115Kgをモレキユラシーブ膜
（ポアサイズ分子量20000）で加圧濾過し、さらに
洗滌して濃縮し12Kgの液を得た。本液を飽和度55
％で硫安塩析を行い、沈殿を遠心分離して回収し
た。これを0.005Mトリス−HC緩衝液（PH7.5）
に溶解し980ｇの液を得た。さらに本液を24時間
透析した。
次にDEAE−アガロースゲルを用いたイオン交
換クロマト（φ44×500mm）により精製し、グル
コアミラーゼ活性を有するフラクシヨン液360ml
を回収した。これをモレキユラシーブ膜で濾過し
濃縮して30単位／mlの酵素液160ｇを得た。さら
に、本液を凍結乾燥し架橋デキユトランゲルのカ
ラムでゲル濾過し、活性フラクシヨン40ｇを得た
（92単位／ml）。
次に、局法馬鈴薯殿粉10ｇと60℃温水90mlとの
混合物に前述の耐熱性α−アミラーゼ2000単位を
添加し80℃で３時間反応し、限界デキストンに転
換した。
次いで、上記の液化処理液に上述のグルコアミ
ラーゼ1000単位を添加し、PH4.5，70℃，３時間
反応して糖化した。
その結果、DE（Dextrose Equivalent Value
DE＝直接還元糖（ぶどう糖として標示）／固形分×100
が95.8の糖化液を得た。
実施例 ２
攪拌機を有する0.3容オートクレーブ内にと
うもろこし殿粉10ｇ、水道水90ｇ及び実施例１で
調製したα−アミラーゼ1000単位を添加し、攪拌
下、まず、オートクレーブ内気相部へゲージ圧２
Kg／cm2の水蒸気を排気弁開放下10秒間吹込み、次
いで、排気弁を閉め、コーンスターチスラリ内へ
蒸気を吹込み、内圧を1.1Kg／cm2に調節し１時間
保持した。次いで液化液をオートクレーブより取
出し、80℃まで冷却したあとα−アミラーゼ1000
単位を加え２時間反応させて液化液を調製した。
上記液化液に実施例１にて調製したグルコアミ
ラーゼ1000単位を加え、70℃で３時間反応した。
その結果、DEが93.2の糖化液を得た。なお糖
化後の反応液のPHは4.8であつた。
実施例 ３
甘しよ殿粉10Kgについて、実施例１の手法によ
り液化及び糖化の反応を行つたところ、DE96.3
の糖化液が得られた。
実施例 ４
局法法鈴薯殿粉10ｇに水道水90mlを加え70℃に
加温した。次いで、実施例１にて調製したα−ア
ミラーゼを2000単位及びグルコアミラーゼ1000単
位を加え、70℃５時間反応させた。反応終了後の
液PHは4.8であつた。反応液のDEは92.8であつた。
第９図は、本発明の糖製造方法を実施する製造
装置の１実施例を示す図である。本実施例では、
殿粉を原料として用いる。攪拌装置、PH調整機構
を有する殿粉スラリ調製槽１１に、水貯留槽１
８、殿粉貯留槽１９より、それぞれ水及び殿粉を
加えて攪拌し、殿粉スラリを調製する。ここで用
いる水としては、カルシウム塩を100μM程度含
むことが望ましい。通常、原料水として工業用
水，飲料用水道水等を用いれば特にカルシウム塩
添加を必要とすることはない。原料の殿粉は特に
限定されるものではなく、馬鈴薯殿粉，とうもろ
こし殿粉，甘しよ殿粉等を用いる。殿粉スラリの
濃度としては10〜40％にすることが望ましい。さ
らにあらかじめ、殿粉スラリを加熱糊化した場合
のPHを測定しておき、糊化後のPHが４〜５による
ようPH調整薬剤貯槽２２よりPH調整薬剤を加え、
調整しておくことが望ましい。しかしながら、通
常の場合、殿粉スラリを糊化するとその糊化液の
PHは４〜５を示すことが多く、薬剤添加を必要と
することは少ない。PH補正を終えた殿粉スラリに
α−アミラーゼ貯槽２１よりα−アミラーゼを加
え、十分に攪拌する。次いで、送液ポンプ３５に
より殿粉スラリをスラリ加熱器１５に移送する。
本スラリ加熱器では、殿粉スラリ中に直接蒸気が
注入され、瞬時に100〜105℃まで加熱され、直ち
に液化槽１６に移送される。液化槽１６では70〜
100℃に10〜60分間保持され、その間、殿粉の糊
化、及びα−アミラーゼによる液化が行われ、液
の粘度が急速に低下する。液化終了後、液化液は
送液ポンプ３８より、糖化槽１３に移送される。
糖化槽１３には攪拌装置、加温機構、PH測定装置
が設置されている。液化液にグルコアミラーゼ貯
槽２３よりグルコアミラーゼが供給され、攪拌
下、70〜80℃にて10〜50時間、酵素反応が行われ
る。すなわち、液化により生成したデキストリン
をグルコースに加水分解する。糖化終了後、糖化
液は送液ポンプ３６により糖化液貯槽１４に移送
し、後段の精製工程に移送するまでの間、貯蔵す
る。精製工程への移送の際には送液ポンプ３７に
より圧送する。
第１０図は、本発明になる糖製造方法を実施す
る製造装置の一実施例を示すフロー図である。本
実施例は、デキストリンを糖化原料としている例
である。原料のデキストリンはデキストリン貯槽
２０よりデキストリン液調整槽１２に供給され
る。さらに水貯留槽８より水を加えて、攪拌装置
３１により攪拌し、ロ形物濃度10〜80％のデキス
トリン溶液を調製する。次いで、デキストリン溶
液のPHを測定し、PH調整用薬剤貯槽２２よりPH調
整用薬剤を加えPH４〜５に調整する。次いで、グ
ルコアミラーゼ貯槽よりグルコアミラーゼをロ形
物当り１×104〜５×105単位加え、攪拌，混合す
る。そののち、送液ポンプ３５により糖化槽１３
に移送する。糖化槽１３は、攪拌装置３２、加温
装置及びPH測定装置を有している。デキストリン
溶液は糖化槽１３内にて70〜80℃に加温され、10
〜50時間保持される間にグルコアミラーゼにより
グルコアミラーゼに加水分解される。糖化終了
後、糖化液は送液ポンプ３６により糖化液貯槽１
４に移送し、貯蔵する。精製工程にあわせて、送
液ポンプ３７により供給する。
〔考案の効果〕
本発明によれば、原料の殿粉カラリーを中和せ
ず酸性のまま高温で反応できる。そのため、高速
で反応できかつ中和剤の添加やカルシウム塩の添
加が不要となり、製品糖液の脱塩工程への負荷が
大巾に軽減される。
To produce glucose or isomerized sugar, the raw material starch is first liquefied with α-amylase and then saccharified with glucoamylase. During liquefaction, 20 to 40% of the raw material starch is Because it is prepared at a high concentration, the pH of the liquid is acidic. For this reason, the conventional α
- When using amylase and glucoamylase, their action PH range is in the neutral range, so they must be neutralized with alkali. On the other hand, the heat-resistant and acid-resistant α-amylase and glucoamylase used in the present invention have high activity even in the acidic range.
Remains acidic without being neutralized by liquefaction (in this case, PH adjustment is not necessarily required)
It is possible to perform liquefaction and saccharification, and as a result, the load on the desalting process after the reaction can be significantly reduced. In addition, since the new glucoamylase used in the present invention has activity even at high temperatures of 80°C or higher,
After being liquefied at a high temperature of °C, it can be used as is without cooling. [Embodiments of the Invention] Hereinafter, embodiments of the present invention will be shown and explained in more detail. Example 1 Soluble starch 1.5%, polypeptone 0.5%, yeast extract 0.5%, monoolium phosphate 0.7%, dibasic sodium phosphate 0.35%, magnesium sulfate dihydrate
Dispense 15.75 kg of a liquid medium (PH6.5) containing 0.01% sodium thioglycolate, 0.1% sodium thioglycolate, and tap water into 5 culture tanks with an internal volume of 5, and incubate at 120°C.
Sterilize for 20 minutes. To each tank was added 350 g of a bacterial cell suspension of the genus Clostridium, which had been anaerobically cultured in the same medium as above and was isolated by the present inventors. Next, a water seal trap is attached to the gas outlet, and after the gas phase inside the fermenter is sufficiently replaced with argon gas, the culture is carried out under air-shielded conditions. The pH of the culture solution is automatically adjusted to 6.0, and the temperature is automatically adjusted to 60℃. After culturing for 22 hours, the cultures were combined and centrifuged at 6000 rpm to remove bacterial cells.
This supernatant showed a specific activity of 60 units/b. Next, 14.6 kg of the above supernatant liquid was cooled to 2° C., and then 300 g of corn starch and 300 g of Celite (manufactured by Wako Pure Chemical Industries, Ltd.) were added and brought into contact with each other for 10 minutes while stirring.
Next, cornstarch and celite were collected by vacuum filtration, and further washed with pure water 3 cooled to 2°C. Next, 0.05M Tris containing 5mM calcium chloride, prewarmed to 65°C,
The above cornstarch and Celite were dispersed in a hydrochloric acid buffer (PH7.5), held at 65°C for 5 minutes on a water bath, solid-liquid was separated by vacuum filtration, and the solids were further dispersed in the above buffer (65°C) in 1. The filtrate and washing solution were combined to obtain 4. Next, the above-mentioned filtration was cooled to 2°C, filtered and concentrated through a molecular sieve membrane (molecular weight cut off: 20,000), and insoluble matter was further removed by filtration to obtain 400 ml of a concentrated liquid. This concentrate showed α-amylase activity of 1.5×10 3 units/ml. Next, the above concentrated solution was added to a diethylaminoethylated cross-linked agarose gel (DEAE Sepharose/CL).
Purification was performed by ion exchange chromatography (column size: φ50 x 210 mm) using a column (Column size: φ50 x 210 mm) using a column (Column size: φ50 x 210 mm). First, the above concentrated solution was dialyzed twice with 0.05M Tris-HCl buffer (PH7.5), and then insoluble materials were filtered and charged to a gel column buffered with the same buffer and washed. The sodium chloride concentration in the buffer was then developed in an increasing linear gradient. As a result, the sodium chloride concentration was 0.04M.
Two peaks with α-amylase activity were observed at the 0.08M position. The amount of α-amylase activity eluted at the 0.04M position is approximately 30% of the total adsorbed activity.
%, that of α-amylase eluted at the 0.08M position was about 60%. Both active fractions were dialyzed in pure water and then lyophilized to obtain α-amylase,
and α-amylase were obtained. The activity recovery rates based on the centrifuged supernatant of the culture solution were 17% and 32%, respectively. Next, dextrin 1.5%, polypeptone 0.5%,
Yeast extract 0.5%, potassium phosphate 0.7%, dibasic sodium phosphate 0.2%, magnesium sulfate.
Heptahydrate 0.001%, Sodium thioglycolate 0.1%
Pour 120 kg of liquid medium (PH6.0) containing water and tap water into 10 culture tanks with an internal volume of 20, and heat at 120℃.
Sterilized for 15 minutes. Clostridium, which is a glucoamylase-producing bacterium according to the present invention, was placed in each culture tank.
Cell suspension of SPG-0005 (Feikoken Bacteria No. 8737)
0.5Kg was added. Next, the gas phase was replaced with nitrogen, and cultured under anaerobic conditions at 60°C and pH 6.0 for 2 days.
The culture solution was centrifuged at 6000 rpm for 10 minutes to remove bacterial cells. The collected supernatant liquid was further filtered through glass filter paper to obtain a solid particle-free culture filtrate. This solution contains 0.05 units/ml of glucoamylase. Next, 115 kg of the above culture filtrate was filtered under pressure using a molecular sieve membrane (pore size, molecular weight 20,000), further washed and concentrated to obtain 12 kg of liquid. This liquid is saturated at 55
% ammonium sulfate salting out, and the precipitate was collected by centrifugation. Add this to 0.005M Tris-HC buffer (PH7.5)
980g of liquid was obtained. Furthermore, this solution was dialyzed for 24 hours. Next, it was purified by ion exchange chromatography (φ44 x 500 mm) using DEAE-agarose gel, and 360 ml of a fraction solution containing glucoamylase activity was purified.
was recovered. This was filtered through a molecular sieve membrane and concentrated to obtain 160 g of an enzyme solution containing 30 units/ml. Furthermore, this solution was freeze-dried and gel-filtered through a cross-linked dexyutran gel column to obtain 40 g of active fraction (92 units/ml). Next, 2000 units of the above-mentioned heat-stable α-amylase were added to a mixture of 10 g of topical potato starch and 90 ml of 60° C. hot water, and the mixture was reacted at 80° C. for 3 hours to convert into limit dextone. Next, 1000 units of the above-mentioned glucoamylase was added to the above-mentioned liquefied solution, and the mixture was reacted at pH 4.5 and 70°C for 3 hours to perform saccharification. As a result, a saccharified solution with a DE (Dextrose Equivalent Value DE=direct reducing sugar (labeled as glucose)/solid content x 100) of 95.8 was obtained. Example 2 10 g of corn starch and tap water were placed in a 0.3 volume autoclave equipped with a stirrer. Add 90 g of α-amylase and 1000 units of α-amylase prepared in Example 1, and while stirring, first apply a gauge pressure of 2 to the gas phase inside the autoclave.
Kg/cm 2 of water vapor was blown into the slurry for 10 seconds with the exhaust valve open, and then the exhaust valve was closed and steam was blown into the cornstarch slurry to adjust the internal pressure to 1.1 Kg/cm 2 and hold for 1 hour. Next, the liquefied liquid was taken out from the autoclave, cooled to 80℃, and then treated with α-amylase 1000
The units were added and reacted for 2 hours to prepare a liquefied liquid. 1000 units of glucoamylase prepared in Example 1 was added to the above liquefied solution, and the mixture was reacted at 70°C for 3 hours. As a result, a saccharified liquid with a DE of 93.2 was obtained. The pH of the reaction solution after saccharification was 4.8. Example 3 When 10 kg of Amashiyo starch was subjected to liquefaction and saccharification reactions using the method of Example 1, DE96.3 was obtained.
A saccharified liquid was obtained. Example 4 90 ml of tap water was added to 10 g of local method bell pepper starch and heated to 70°C. Next, 2000 units of α-amylase prepared in Example 1 and 1000 units of glucoamylase were added and reacted at 70°C for 5 hours. After the reaction was completed, the liquid pH was 4.8. The DE of the reaction solution was 92.8. FIG. 9 is a diagram showing one embodiment of a manufacturing apparatus for carrying out the sugar manufacturing method of the present invention. In this example,
Starch is used as a raw material. A starch slurry preparation tank 11 with a stirring device and a PH adjustment mechanism, and a water storage tank 1
8. Add water and starch from the starch storage tank 19 and stir to prepare starch slurry. The water used here desirably contains about 100 μM of calcium salt. Usually, if industrial water, drinking tap water, etc. are used as raw water, there is no need to add calcium salts. The raw material starch is not particularly limited, and potato starch, corn starch, amashiyo starch, etc. are used. The concentration of starch slurry is preferably 10 to 40%. Furthermore, the PH when the starch slurry is heated and gelatinized is measured in advance, and a PH adjustment agent is added from the PH adjustment agent storage tank 22 so that the PH after gelatinization is 4 to 5.
It is advisable to adjust it. However, in normal cases, when starch slurry is gelatinized, the gelatinization liquid is
The pH often shows a value of 4 to 5, and drug addition is rarely required. α-Amylase is added from the α-amylase storage tank 21 to the starch slurry that has undergone pH correction, and is thoroughly stirred. Next, the starch slurry is transferred to the slurry heater 15 by the liquid transfer pump 35.
In this slurry heater, steam is directly injected into the starch slurry, the slurry is instantaneously heated to 100 to 105°C, and the slurry is immediately transferred to the liquefaction tank 16. 70~ for liquefaction tank 16
The temperature is maintained at 100°C for 10 to 60 minutes, during which time the starch is gelatinized and liquefied by α-amylase, resulting in a rapid decrease in the viscosity of the liquid. After the liquefaction is completed, the liquefied liquid is transferred to the saccharification tank 13 by the liquid sending pump 38.
The saccharification tank 13 is equipped with a stirring device, a heating mechanism, and a PH measuring device. Glucoamylase is supplied to the liquefied liquid from the glucoamylase storage tank 23, and an enzymatic reaction is performed at 70 to 80°C for 10 to 50 hours while stirring. That is, dextrin produced by liquefaction is hydrolyzed into glucose. After the saccharification is completed, the saccharified liquid is transferred to the saccharified liquid storage tank 14 by the liquid sending pump 36 and stored until it is transferred to the subsequent purification process. When transferring to the purification process, the liquid is pumped by the liquid feeding pump 37. FIG. 10 is a flow diagram showing an embodiment of a manufacturing apparatus for carrying out the sugar manufacturing method of the present invention. This example is an example in which dextrin is used as the raw material for saccharification. Dextrin as a raw material is supplied from a dextrin storage tank 20 to a dextrin liquid adjustment tank 12 . Furthermore, water is added from the water storage tank 8 and stirred by the stirring device 31 to prepare a dextrin solution having a lozenge concentration of 10 to 80%. Next, the pH of the dextrin solution is measured, and a pH adjusting agent is added from the pH adjusting agent storage tank 22 to adjust the pH to 4 to 5. Next, 1×10 4 to 5×10 5 units of glucoamylase are added per cube from the glucoamylase storage tank, and the mixture is stirred and mixed. After that, the saccharification tank 13 is
Transfer to. The saccharification tank 13 has a stirring device 32, a heating device, and a PH measuring device. The dextrin solution is heated to 70 to 80°C in the saccharification tank 13, and
It is hydrolyzed to glucoamylase by glucoamylase during holding for ~50 hours. After the completion of saccharification, the saccharified liquid is transferred to the saccharified liquid storage tank 1 by the liquid sending pump 36.
4 and store. The liquid is supplied by a liquid feeding pump 37 in accordance with the purification process. [Effects of the invention] According to the present invention, the starch curry as a raw material can be reacted at high temperature while being acidic without being neutralized. Therefore, the reaction can be carried out at high speed, and there is no need to add a neutralizing agent or calcium salt, and the load on the process of desalting the product sugar solution is greatly reduced.
第１図は本発明に用いるα−アミラーゼのPH特
性図、第２図は本α−アミラーゼの温度特性図、
第３図は本α−アミラーゼの耐熱性を示す特性
図、第４図は本α−アミラーゼのカルシウム添加
による耐熱性向上を示す特性図、第５図は、本発
明に用いるグルコアミラーゼのPH特性図、第６図
は本グルコアミラーゼのPH安定性を示す特性図、
第７図は本グルコアミラーゼの温度特性図、第８
図は本グルコアミラーゼの耐熱性を示す特性図で
ある。第９図は本発明の糖製造方法を実施する製
造装置の一実施例を示すフロー図、第１０図は他
の実施例を示すフロー図である。
１……本発明α−アミラーゼ、２……本発明
α−アミラーゼ、３……バチルス・ステアロサ
ーモフイラス起源α−アミラーゼ、４……バチル
ス・スブチリス起源α−フミラーゼ、５……バチ
ルス・リチエニホルミス起源α−アミラーゼ、６
……バチルス・リチエニホルミス起源α−アミラ
ーゼ、７……バチルス・リチエニホルミスspを起
源とする耐熱性α−アミラーゼ、８……バチル
ス・ズブチリスspを起源とするα−アミラーゼ、
１１……殿粉スラリ調製槽、１２……デキストリ
ン液調製糖、１３……糖化槽、１４……糖化液貯
槽、１５……スラリ加熱器、１６……液化槽、１
８……水貯留槽、１９……殿粉貯槽、２０……デ
キストリン貯槽、２１……αアミラーゼ貯槽、２
２……PH調整薬剤貯槽、３１，３２，３３，３４
……攪拌装置、３５，３６，３７，３８……送液
ポンプ。
Figure 1 is a PH characteristic diagram of α-amylase used in the present invention, Figure 2 is a temperature characteristic diagram of this α-amylase,
Figure 3 is a characteristic diagram showing the heat resistance of the present α-amylase, Figure 4 is a characteristic diagram showing the improvement in heat resistance of the present α-amylase due to the addition of calcium, and Figure 5 is the PH characteristic of the glucoamylase used in the present invention. Figure 6 is a characteristic diagram showing the PH stability of this glucoamylase.
Figure 7 is a temperature characteristic diagram of this glucoamylase, Figure 8
The figure is a characteristic diagram showing the heat resistance of the present glucoamylase. FIG. 9 is a flowchart showing one embodiment of a manufacturing apparatus for carrying out the sugar manufacturing method of the present invention, and FIG. 10 is a flowchart showing another embodiment. 1... alpha-amylase of the present invention, 2... alpha-amylase of the present invention, 3... alpha-amylase originating from Bacillus stearothermophilus, 4... alpha-humylase originating from Bacillus subtilis, 5... originating from Bacillus lithieniformis α-amylase, 6
...α-amylase originating from Bacillus lithieniformis, 7...Thermostable α-amylase originating from Bacillus lithieniformis sp, 8...α-amylase originating from Bacillus subtilis sp,
11... Starch slurry preparation tank, 12... Dextrin liquid preparation sugar, 13... Saccharification tank, 14... Saccharification liquid storage tank, 15... Slurry heater, 16... Liquefaction tank, 1
8...Water storage tank, 19...Starch storage tank, 20...Dextrin storage tank, 21...α-amylase storage tank, 2
2...PH adjustment drug storage tank, 31, 32, 33, 34
... Stirring device, 35, 36, 37, 38 ... Liquid pump.
Claims (1)
グルコアミラーゼを用いて生成したデキストリン
を糖化する殿粉の糖化方法において、原料殿粉を
けん濁した液に前記α−アミラーゼとしてクロス
ツリジウム属に属する微工研菌寄第7918号の好熱
性嫌気性細菌より産生した耐熱性α−アミラーゼ
を添加し、金属イオン無添加でかつ原料殿粉けん
濁液のPHを調整することなく酸性のままで殿粉を
液化し、生成したデキストリンをクロスツリジウ
ム属に属する微工研菌寄第8737号の好熱性嫌気性
細菌より産生した耐熱性グルコアミラーゼを用い
てPHを調整することなく酸性のままで糖化するこ
とを特徴とする糖の製造方法。1 After liquefying the starch using α-amylase,
In a starch saccharification method in which dextrin produced using glucoamylase is saccharified, thermophilic anaerobic anaerobic strain No. 7918 belonging to the genus Clostridium is added to a suspension of raw material starch as the α-amylase. By adding heat-stable α-amylase produced from sex bacteria, the starch is liquefied without adding metal ions and without adjusting the pH of the raw starch suspension, and the resulting dextrin is liquefied with Clostridium. A method for producing sugar characterized by saccharifying the sugar while keeping it acidic without adjusting the PH using a thermostable glucoamylase produced from a thermophilic anaerobic bacterium belonging to the genus Microtechnological Laboratory No. 8737.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61185129A JPS6342695A (en) | 1986-08-08 | 1986-08-08 | Method for producing sugar and production plant therefor |
| EP87111005A EP0255124A3 (en) | 1986-07-29 | 1987-07-29 | Thermostable glucoamylase, a method for production of glucose using same and a plant for production thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61185129A JPS6342695A (en) | 1986-08-08 | 1986-08-08 | Method for producing sugar and production plant therefor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6342695A JPS6342695A (en) | 1988-02-23 |
| JPH0428356B2 true JPH0428356B2 (en) | 1992-05-14 |
Family
ID=16165379
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61185129A Granted JPS6342695A (en) | 1986-07-29 | 1986-08-08 | Method for producing sugar and production plant therefor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6342695A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5375342A (en) * | 1976-12-13 | 1978-07-04 | Cpc International Inc | Production of glucose by immobilised glucoamylase |
| JPS57132893A (en) * | 1981-02-09 | 1982-08-17 | Denki Kagaku Kogyo Kk | Preparation of glucose |
| JPS6041482A (en) * | 1983-07-13 | 1985-03-05 | シ−・ピ−・シ−・インタ−ナシヨナル・インコ−ポレイテツド | New heat stable acid resistance alpha-amylase and its preparation |
| JPS6054680A (en) * | 1983-08-17 | 1985-03-29 | シ−・ピ−・シ−・インタ−ナシヨナル・インコ−ポレイテツド | Novel heat-stable glucoamylase and its production |
| JPS61115496A (en) * | 1984-11-09 | 1986-06-03 | Hitachi Ltd | Method of liquefying starch |
-
1986
- 1986-08-08 JP JP61185129A patent/JPS6342695A/en active Granted
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5375342A (en) * | 1976-12-13 | 1978-07-04 | Cpc International Inc | Production of glucose by immobilised glucoamylase |
| JPS57132893A (en) * | 1981-02-09 | 1982-08-17 | Denki Kagaku Kogyo Kk | Preparation of glucose |
| JPS6041482A (en) * | 1983-07-13 | 1985-03-05 | シ−・ピ−・シ−・インタ−ナシヨナル・インコ−ポレイテツド | New heat stable acid resistance alpha-amylase and its preparation |
| JPS6054680A (en) * | 1983-08-17 | 1985-03-29 | シ−・ピ−・シ−・インタ−ナシヨナル・インコ−ポレイテツド | Novel heat-stable glucoamylase and its production |
| JPS61115496A (en) * | 1984-11-09 | 1986-06-03 | Hitachi Ltd | Method of liquefying starch |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6342695A (en) | 1988-02-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4284722A (en) | Heat and acid-stable alpha-amylase enzymes and processes for producing the same | |
| JPH04503757A (en) | Novel hyperthermally stable α-amylase | |
| JPS5953038B2 (en) | Manufacturing method of cyclodextrin | |
| EP0135138A2 (en) | A novel thermostable glucoamylase and method for its production | |
| JPS6225036B2 (en) | ||
| JPH0365156B2 (en) | ||
| US4647538A (en) | Thermostable beta-amylase | |
| EP0184019B1 (en) | Thermostable alpha-amylase-producing, thermophilic anaerobic bacteria, thermostable alpha-amylase and process for producing the same | |
| JPH0118717B2 (en) | ||
| FI58157C (en) | VAERME- OCH SYRASTABILT ALFA-AMYLAS-ENZYM OCH FOERFARANDE FOER DESS FRAMSTAELLNING | |
| US5281527A (en) | Process for producing pullalanase | |
| JPS62283988A (en) | Production of maltooligosaccharide | |
| EP0255124A2 (en) | Thermostable glucoamylase, a method for production of glucose using same and a plant for production thereof | |
| JPH0428356B2 (en) | ||
| JPH05292962A (en) | New branch-cleaving enzyme and its production | |
| JPH0365149B2 (en) | ||
| JPH0425794B2 (en) | ||
| JPS6342696A (en) | Production of sugar | |
| JPH0324197B2 (en) | ||
| KR960007741B1 (en) | Novel(alpha)-1,6-glucosidase and process for producing the same | |
| JPS6219081A (en) | Production of heat-resistant alpha-amylase | |
| JPH047672B2 (en) | ||
| CN116064480A (en) | Alpha-glucosidase cyclization mutant with improved thermal stability and construction method thereof | |
| SU1449583A1 (en) | Strain of bacillus subtilis bacteria as producer of restriction endonuclease bsu 151 | |
| JPS6225037B2 (en) |