JP2008271826A - Method for producing cellulase - Google Patents
Method for producing cellulase Download PDFInfo
- Publication number
- JP2008271826A JP2008271826A JP2007118021A JP2007118021A JP2008271826A JP 2008271826 A JP2008271826 A JP 2008271826A JP 2007118021 A JP2007118021 A JP 2007118021A JP 2007118021 A JP2007118021 A JP 2007118021A JP 2008271826 A JP2008271826 A JP 2008271826A
- Authority
- JP
- Japan
- Prior art keywords
- cellulase
- cellulose
- producing
- lactose
- raw material
- 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.)
- Granted
Links
- 108010059892 Cellulase Proteins 0.000 title claims abstract description 84
- 229940106157 cellulase Drugs 0.000 title claims abstract description 81
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 17
- 239000001913 cellulose Substances 0.000 claims abstract description 63
- 238000000034 method Methods 0.000 claims abstract description 42
- 229920002678 cellulose Polymers 0.000 claims abstract description 35
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims abstract description 26
- 239000008101 lactose Substances 0.000 claims abstract description 26
- 239000002994 raw material Substances 0.000 claims abstract description 18
- 244000005700 microbiome Species 0.000 claims abstract description 16
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 235000010980 cellulose Nutrition 0.000 claims description 30
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 28
- 229920003124 powdered cellulose Polymers 0.000 claims description 28
- 235000019814 powdered cellulose Nutrition 0.000 claims description 28
- 241000894006 Bacteria Species 0.000 claims description 22
- 239000010902 straw Substances 0.000 claims description 13
- 241001019659 Acremonium <Plectosphaerellaceae> Species 0.000 claims description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 9
- 241000209094 Oryza Species 0.000 claims description 9
- 235000007164 Oryza sativa Nutrition 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- 235000009566 rice Nutrition 0.000 claims description 9
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 8
- 241001215623 Talaromyces cellulolyticus Species 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- -1 Avicel Substances 0.000 claims description 5
- 239000005862 Whey Substances 0.000 claims description 5
- 102000007544 Whey Proteins Human genes 0.000 claims description 5
- 108010046377 Whey Proteins Proteins 0.000 claims description 5
- 230000007062 hydrolysis Effects 0.000 claims description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims description 5
- 241000609240 Ambelania acida Species 0.000 claims description 4
- 239000010905 bagasse Substances 0.000 claims description 4
- 239000010893 paper waste Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 239000002023 wood Substances 0.000 claims description 4
- 235000013365 dairy product Nutrition 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- WSVLPVUVIUVCRA-KPKNDVKVSA-N Alpha-lactose monohydrate Chemical compound O.O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O WSVLPVUVIUVCRA-KPKNDVKVSA-N 0.000 claims description 2
- 241000209140 Triticum Species 0.000 claims description 2
- 235000021307 Triticum Nutrition 0.000 claims description 2
- 239000000758 substrate Substances 0.000 abstract description 10
- 239000001963 growth medium Substances 0.000 abstract description 3
- 230000000593 degrading effect Effects 0.000 abstract 3
- 239000002609 medium Substances 0.000 description 19
- 108090000790 Enzymes Proteins 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 16
- 229940088598 enzyme Drugs 0.000 description 16
- 239000000243 solution Substances 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 239000000123 paper Substances 0.000 description 9
- 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 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000006911 enzymatic reaction Methods 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 241000228212 Aspergillus Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000228143 Penicillium Species 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- WDMUXYQIMRDWRC-UHFFFAOYSA-N 2-hydroxy-3,4-dinitrobenzoic acid Chemical compound OC(=O)C1=CC=C([N+]([O-])=O)C([N+]([O-])=O)=C1O WDMUXYQIMRDWRC-UHFFFAOYSA-N 0.000 description 2
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 241000223259 Trichoderma Species 0.000 description 2
- 241000223261 Trichoderma viride Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 150000002016 disaccharides Chemical class 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 229930182478 glucoside Natural products 0.000 description 2
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical compound NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000003094 microcapsule Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- AVTYONGGKAJVTE-OLXYHTOASA-L potassium L-tartrate Chemical compound [K+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O AVTYONGGKAJVTE-OLXYHTOASA-L 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- MWBWWFOAEOYUST-UHFFFAOYSA-N 2-aminopurine Chemical compound NC1=NC=C2N=CNC2=N1 MWBWWFOAEOYUST-UHFFFAOYSA-N 0.000 description 1
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 1
- KKAJSJJFBSOMGS-UHFFFAOYSA-N 3,6-diamino-10-methylacridinium chloride Chemical compound [Cl-].C1=C(N)C=C2[N+](C)=C(C=C(N)C=C3)C3=CC2=C1 KKAJSJJFBSOMGS-UHFFFAOYSA-N 0.000 description 1
- YHQDZJICGQWFHK-UHFFFAOYSA-N 4-nitroquinoline N-oxide Chemical compound C1=CC=C2C([N+](=O)[O-])=CC=[N+]([O-])C2=C1 YHQDZJICGQWFHK-UHFFFAOYSA-N 0.000 description 1
- LQLQRFGHAALLLE-UHFFFAOYSA-N 5-bromouracil Chemical compound BrC1=CNC(=O)NC1=O LQLQRFGHAALLLE-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 108010084185 Cellulases Proteins 0.000 description 1
- 102000005575 Cellulases Human genes 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- OWIKHYCFFJSOEH-UHFFFAOYSA-N Isocyanic acid Chemical class N=C=O OWIKHYCFFJSOEH-UHFFFAOYSA-N 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- WDVSHHCDHLJJJR-UHFFFAOYSA-N Proflavine Chemical compound C1=CC(N)=CC2=NC3=CC(N)=CC=C3C=C21 WDVSHHCDHLJJJR-UHFFFAOYSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- 241000499912 Trichoderma reesei Species 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000012445 acidic reagent Substances 0.000 description 1
- 229940023020 acriflavine Drugs 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000002168 alkylating agent Substances 0.000 description 1
- 229940100198 alkylating agent Drugs 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 238000006149 azo coupling reaction Methods 0.000 description 1
- 150000007514 bases Chemical class 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- FYGDTMLNYKFZSV-ZWSAEMDYSA-N cellotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-ZWSAEMDYSA-N 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- UGWKCNDTYUOTQZ-UHFFFAOYSA-N copper;sulfuric acid Chemical compound [Cu].OS(O)(=O)=O UGWKCNDTYUOTQZ-UHFFFAOYSA-N 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- WMPOZLHMGVKUEJ-UHFFFAOYSA-N decanedioyl dichloride Chemical compound ClC(=O)CCCCCCCCC(Cl)=O WMPOZLHMGVKUEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-O guanidinium Chemical compound NC(N)=[NH2+] ZRALSGWEFCBTJO-UHFFFAOYSA-O 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 239000002029 lignocellulosic biomass Substances 0.000 description 1
- 229960003390 magnesium sulfate Drugs 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
- 150000004706 metal oxides Chemical class 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920006254 polymer film Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920005990 polystyrene resin Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 239000005373 porous glass Substances 0.000 description 1
- 239000001472 potassium tartrate Substances 0.000 description 1
- 229940111695 potassium tartrate Drugs 0.000 description 1
- 235000011005 potassium tartrates Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 229960000286 proflavine Drugs 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 101150089778 pyr-4 gene Proteins 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 238000001223 reverse osmosis Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 description 1
- 229940120668 salicin Drugs 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229940047022 zinc sulfate 10 mg Drugs 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)
Abstract
Description
本発明は、セルロース製造用基質、該基質を用いたセルラーゼの製造方法、およびセルロースの分解または糖化方法に関する。 The present invention relates to a substrate for producing cellulose, a method for producing cellulase using the substrate, and a method for decomposing or saccharifying cellulose.
セルラーゼとは、セルロースを、グルコース、セロビオースおよびセロオリゴ糖に加水分解する酵素反応系を触媒する酵素群の総称であり、その作用様式により、エキソ−β−グルカナーゼ、エンド−β−グルカナーゼおよびβ−グルコシダーゼなどに分類される。セルラーゼのこれら酵素の相互作用により、セルロースが最終的にグルコースまで分解される（特許文献１）。 Cellulase is a general term for an enzyme group that catalyzes an enzyme reaction system that hydrolyzes cellulose into glucose, cellobiose, and cellooligosaccharide. Depending on its mode of action, exo-β-glucanase, endo-β-glucanase, and β-glucosidase And so on. Cellulase is finally decomposed into glucose by the interaction of these enzymes of cellulase (Patent Document 1).
一方、近年、セルラーゼを用いて、リグノセルロース系バイオマス資源を酵素分解、糖化することによって構成単位であるグルコース、キシロースにし、更にこれを発酵することによって得られるエタノールや乳酸などを液体燃料もしくは化学原料として利用することが検討されている。糖化に用いるセルラーゼの製造には従来、セルラーゼ生産菌としてアクレモニウム・セルロリティカス（Acremonium cellulolyticus）（特許文献１）、トリコデルマ・レーゼイ（Trichoderma reesei）（非特許文献１）、トリコデルマ・ビリデ（T. viride）（非特許文献２）やアスペルギルス（Aspergillus）属、ペニシリウム（Penicillium）属等に属する微生物が使われており、そして炭素源としては、セルロースを含有する原料が基質として用いられている（特許文献１）。しかしながら、これらの方法は十分なセルラーゼ生産性が得られないという欠点があった。 On the other hand, in recent years, lignocellulosic biomass resources using cellulase are enzymatically decomposed and saccharified to form constituent units such as glucose and xylose, and ethanol, lactic acid, etc. obtained by fermenting them are used as liquid fuels or chemical raw materials. It is being considered to be used. Cellulase used for saccharification has been conventionally produced as cellulase-producing bacteria, Acremonium cellulolyticus (Patent Document 1), Trichoderma reesei (Non-Patent Document 1), Trichoderma Viride (T. viride) (Non-Patent Document 2), microorganisms belonging to the genus Aspergillus, Penicillium, etc. are used, and as a carbon source, a raw material containing cellulose is used as a substrate (patent) Reference 1). However, these methods have a drawback that sufficient cellulase productivity cannot be obtained.
本発明の課題は、セルラーゼの生産性を高める、セルラーゼ生産菌の培養手段を提供し、これにより、セルラーゼの製造、ならびにセルロースの分解、および糖化を、効率的に行うことを目的とする。 An object of the present invention is to provide a means for culturing cellulase-producing bacteria that enhances the productivity of cellulase, and thereby aims to efficiently produce cellulase and decompose and saccharify cellulose.
本発明者らは、上記課題を解決するために鋭意研究した結果、粉末セルロースにラクトースあるいはラクトースを含有する原料を添加して、これを培地としてセルラーゼ生産菌を培養すると、セルラーゼの生産性が著しく向上し、セルラーゼを効率的に製造できるとともに、セルロースの分解、糖化も、安価、効率的に行いうることを見いだし、本発明を完成させた。 As a result of diligent research to solve the above-mentioned problems, the inventors of the present invention added lactose or a raw material containing lactose to powdered cellulose, and cultured cellulase-producing bacteria using this as a medium, the cellulase productivity was remarkably increased. As a result, it was found that cellulase can be efficiently produced, and that cellulose can be decomposed and saccharified at low cost and efficiently, and the present invention has been completed.
本発明は、以下の特徴を有する。
（１）炭素源としてセルロースを含有する原料とラクトースを含有する原料とを含む培地中にてセルラーゼ生産菌を培養し、セルラーゼを製造する方法。
（２）セルロースを含有する原料が、粉末セルロース（アビセルを含む）、古紙類、瀘紙、一般紙類、木材、麦わら、稲わら、ふすま、もみがら、バガス、加水分解残渣、またはそれらの混合物を含む、（１）に記載のセルラーゼの製造方法。
（３）ラクトースを含有する原料が、ラクトース、ラクトース水和物、乳清（ホエイ）、乳製品、またはそれらの混合物を含む、（１）または（２）に記載のセルラーゼの製造方法。
（４）培養が、液体培養または固体培養である、（１）〜（３）のいずれかに記載のセルラーゼの製造方法。
（５）セルラーゼ生産菌がアクレモニウム属に属する微生物である（１）〜（４）のいずれかに記載のセルラーゼの製造方法。
（６）アクレモニウム属に属する微生物がアクレモニウム・セルロリティカス（Acremonium cellulolyticus）Ｃ１株（ＦＥＲＭ Ｐ−１８５０８）である（５）に記載のセルラーゼの製造方法。
（７）（１）〜（４）のいずれかに記載の方法により製造し、得られたセルラーゼを用いてセルロースを分解または糖化することを特徴とするセルロースの分解または糖化方法。
（８）セルラーゼ生産菌がアクレモニウム属に属する微生物である（７）に記載のセルロースの分解または糖化方法。
（９）アクレモニウム属に属する微生物がアクレモニウム・セルロリティカス（Acremonium cellulolyticus）Ｃ１株（ＦＥＲＭ Ｐ−１８５０８）である（８）に記載のセルロースの分解または糖化方法。
The present invention has the following features.
(1) A method for producing cellulase by culturing cellulase-producing bacteria in a medium containing a raw material containing cellulose as a carbon source and a raw material containing lactose.
(2) The raw material containing cellulose is powdered cellulose (including Avicel), waste paper, straw paper, general paper, wood, straw, rice straw, bran, rice bran, bagasse, hydrolysis residue, or a mixture thereof The method for producing cellulase according to (1), comprising:
(3) The method for producing cellulase according to (1) or (2), wherein the raw material containing lactose includes lactose, lactose hydrate, whey (whey), dairy product, or a mixture thereof.
(4) The method for producing cellulase according to any one of (1) to (3), wherein the culture is liquid culture or solid culture.
(5) The method for producing cellulase according to any one of (1) to (4), wherein the cellulase-producing bacterium is a microorganism belonging to the genus Acremonium.
(6) The method for producing cellulase according to (5), wherein the microorganism belonging to the genus Acremonium is Acremonium cellulolyticus C1 strain (FERM P-18508).
(7) A method for decomposing or saccharifying cellulose, comprising decomposing or saccharifying cellulose using the cellulase produced by the method according to any one of (1) to (4).
(8) The method for decomposing or saccharifying cellulose according to (7), wherein the cellulase-producing bacterium is a microorganism belonging to the genus Acremonium.
(9) The method for decomposing or saccharifying cellulose according to (8), wherein the microorganism belonging to the genus Acremonium is Acremonium cellulolyticus C1 strain (FERM P-18508).
本発明は、培地に粉末セルロースおよびラクトースまたはラクトースを含有する原料を添加して、これを用いてセルラーゼ生産菌を培養することによって、セルラーゼの生産性を著しく向上させることができるという格別の作用効果を有する。 In the present invention, powdered cellulose and lactose or a raw material containing lactose are added to a medium, and cellulase-producing bacteria are cultured using the same, whereby cellulase productivity can be remarkably improved. Have
以下に本発明をさらに具体的に説明する。
セルロースは、グルコースがβ−１，４グルコシド結合により高度に重合したグルコースポリマーであり、全ての植物の細胞壁構成成分として存在する。本発明において、「セルロースを含有する原料」は、例えば、粉末セルロース（例えば、Ｓｏｌｋａ Ｆｌｏｃ（登録商標）CAS9004-34-6, Fiber Sales & Development Co. Urbana, OH、およびアビセルなど）、古紙類、瀘紙、一般紙類、木材、麦わら、稲わら、ふすま、もみがら、バガス、加水分解残渣等を含むが、セルラーゼ生産菌の培養のための炭素源となり得る限り、これらに限定されない。加水分解残渣とは、セルロースを含有する原料を、酸や酵素などを用いて加水分解処理したものをいう。本発明において、使用するセルロースを含有する原料としては、上記のものが例示されるが、粉末セルロースが好ましく、特にＳｏｌｋａ Ｆｌｏｃ（登録商標）が好ましい。
The present invention will be described more specifically below.
Cellulose is a glucose polymer in which glucose is highly polymerized by β-1,4 glucoside bonds, and is present as a cell wall constituent of all plants. In the present invention, the “raw material containing cellulose” includes, for example, powdered cellulose (for example, Solka Floc (registered trademark) CAS9004-34-6, Fiber Sales & Development Co. Urbana, OH, and Avicel), waste paper, Including straw paper, general paper, wood, wheat straw, rice straw, bran, rice bran, bagasse, hydrolysis residue, etc., it is not limited to these as long as it can be a carbon source for culturing cellulase-producing bacteria. A hydrolysis residue means what hydrolyzed the raw material containing a cellulose using an acid, an enzyme, etc. In the present invention, examples of the raw material containing cellulose to be used include those described above, and powdered cellulose is preferable, and Solka Floc (registered trademark) is particularly preferable.
ラクトースは、Ｄ−ガラクトースとＤ−グルコースがβ−１，４グルコシド結合した二糖である。哺乳類の乳汁に含まれていることが知られている。本発明において、「ラクトースを含有する原料」としては、例えば、ラクトース（水和物を含む）、乳清（ホエイ）、乳製品等が挙げられるが、これらに限定されない。 Lactose is a disaccharide in which D-galactose and D-glucose are linked by β-1,4 glucoside. It is known to be contained in mammalian milk. In the present invention, examples of the “raw material containing lactose” include, but are not limited to, lactose (including hydrate), whey (whey), and dairy products.
セルラーゼは、上記のようにセルロースを分解する酵素の総称であり、セルロースの分解活性を有していれば、本発明のセルラーゼに含まれる。 Cellulase is a general term for enzymes that decompose cellulose as described above, and is included in the cellulase of the present invention as long as it has cellulolytic activity.
セルラーゼ生産菌は、セルラーゼを生産できる微生物であればいずれでも良く、すなわち、天然源からの分離菌、その変異株、遺伝子組換え菌などを含む。変異株は、紫外線もしくは放射線による照射処理、化学物質（例えば、亜硝酸、塩基類縁化合物（５−ブロモウラシルおよび２−アミノプリンなど）、アルキル化剤（ニトロソグアニジンおよびエチルメタンサルホネートなど）、アクリジン色素類（アクリフラビンおよびプロフラビンなど）、発がん剤（４−ニトロキノリン−１−オキシド）、抗生物質（マイトマイシンＣなど））による処理を行い、処理ずみの菌株からセルラーゼ誘導能の高い菌株を選択することによって得ることができる。遺伝子組換え菌は、公知の手法を用いて作製できる。例えば、菌体を、乳鉢などを用いて液体窒素中で摩粋後、例えばフェノール/クロロホルム法、グアニジウム法又はフェノール/SDS法により全RNAを抽出する。必要に応じて、RNAをオリゴ（ｄT）セルロースカラムに通してpoly(A)+RNAを得るか、あるいは、poly(A)+RNAから既知の逆転写反応を経てcDNAを作製する。NCBI、GenBank等のデータバンクにアクセスすることによって入手可能であるセルラーゼの公知塩基配列を基にプライマーを設計し、上記RNAまたはcDNAを鋳型としてＰＣＲを行い菌体のセルラーゼ遺伝子をクローニングする。これを高発現誘導可能なプロモーター（例えば、pyr4プロモーター、cbhlプロモーターなど）や分泌シグナルペプチドの下流に連結し、宿主微生物と適合性を有する適宜なベクターに組み入れ、遺伝子組換えベクターを作製する。当該ベクターは、ターミネーター領域、抗生物質耐性遺伝子等の選抜用の各種マーカー遺伝子等をさらに含む。あるいは、プライマーを設計する際に、その塩基配列に予め変異を加えておき、そのプライマーを用いて、上記同様ＰＣＲを行うことによって、変異を有するセルラーゼ遺伝子をクローニングすることができる。この変異を有するセルラーゼ遺伝子を用いて遺伝子組換えベクターを作製しても良い。この遺伝子組み換えベクターを、公知の各種遺伝子導入法、例えばカルシウム処理法、遺伝子注入（トランスフェクション）法、パーティクルガン法、エレクトロポレーション法等を用いて宿主微生物へと導入し、変異株を得ることができる。宿主微生物としては、例えば、アクレモニウム属、トリコデルマ属、アスペルギルス属、ペニシリウム属に属するものが挙げられるが、これらに限定されない。なお、上記遺伝子組換えに用いられる分子生物学的手法は、「分子生物学実験プロトコールI, II, III」丸善株式会社発行（１９９７）に記載される方法を参考にして行うことができる。 The cellulase-producing bacterium may be any microorganism that can produce cellulase, ie, an isolated bacterium from a natural source, a mutant thereof, a genetically modified bacterium, and the like. Mutants can be irradiated with ultraviolet rays or radiation, chemical substances (for example, nitrous acid, basic compounds (such as 5-bromouracil and 2-aminopurine), alkylating agents (such as nitrosoguanidine and ethylmethanesulfonate), acridine Treatment with pigments (such as acriflavine and proflavine), carcinogens (4-nitroquinoline-1-oxide), antibiotics (such as mitomycin C)), and select strains with high cellulase-inducing ability from treated strains Can be obtained. The genetically modified bacterium can be prepared using a known technique. For example, after microbial cells are ground in liquid nitrogen using a mortar or the like, total RNA is extracted by, for example, the phenol / chloroform method, the guanidinium method, or the phenol / SDS method. If necessary, RNA is passed through an oligo (dT) cellulose column to obtain poly (A) + RNA, or cDNA is prepared from poly (A) + RNA through a known reverse transcription reaction. Primers are designed based on the known base sequences of cellulases that can be obtained by accessing data banks such as NCBI and GenBank, and PCR is performed using the RNA or cDNA as a template to clone the cellulase gene of the cells. This is ligated downstream of a promoter capable of inducing high expression (eg, a pyr4 promoter, cbhl promoter, etc.) or a secretory signal peptide, and incorporated into an appropriate vector compatible with the host microorganism to prepare a gene recombination vector. The vector further includes various marker genes for selection such as a terminator region and an antibiotic resistance gene. Alternatively, when designing a primer, a cellulase gene having a mutation can be cloned by adding a mutation to the base sequence in advance and performing PCR in the same manner as described above. A gene recombination vector may be prepared using a cellulase gene having this mutation. This recombinant vector is introduced into a host microorganism using various known gene transfer methods such as calcium treatment method, gene injection (transfection) method, particle gun method, electroporation method, etc. to obtain a mutant strain Can do. Examples of host microorganisms include, but are not limited to, those belonging to the genera Acremonium, Trichoderma, Aspergillus, and Penicillium. The molecular biological technique used for the gene recombination can be performed with reference to the method described in “Molecular Biology Experiment Protocols I, II, III” published by Maruzen Co., Ltd. (1997).
セルラーゼ生産菌としては、例えば、アクレモニウム属、トリコデルマ属、アスペルギルス属、ペニシリウム属に属するものが挙げられるが、これらに限定されない。本発明においては、アクレモニウム属に属する微生物が好ましく、特にアクレモニウム・セルロリティカス（Acremonium cellulolyticus）Ｃ１株（ＦＥＲＭ Ｐ−１８５０８）が好ましい。 Examples of cellulase-producing bacteria include, but are not limited to, those belonging to the genus Acremonium, Trichoderma, Aspergillus, and Penicillium. In the present invention, microorganisms belonging to the genus Acremonium are preferable, and Acremonium cellulolyticus C1 strain (FERM P-18508) is particularly preferable.
本発明において「培養」方法としては、液体培養および固体培養が挙げられるが、選択した微生物を培養できるかぎり、これらの方法に限定されない。 In the present invention, “culture” methods include liquid culture and solid culture, but are not limited to these methods as long as the selected microorganism can be cultured.
本発明において、「セルロースを分解または糖化すること」とは、セルロースを分解し、オリゴ糖類、二糖類、単糖類およびそれらの混合物にすることをいう。あるいは、言い換えれば、この用語は、セルラーゼによって、多糖類のグリコシド結合を加水分解することをいう。 In the present invention, “decomposing or saccharifying cellulose” refers to decomposing cellulose into oligosaccharides, disaccharides, monosaccharides, and mixtures thereof. Alternatively, in other words, the term refers to hydrolysis of a polysaccharide glycosidic bond by cellulase.
（セルラーゼ生産菌の培養）
セルラーゼ生産菌は、下記の実施例において具体的に記載されるように培養することが可能である。
(Culture of cellulase-producing bacteria)
Cellulase-producing bacteria can be cultured as specifically described in the examples below.
培地は、炭素源として、粉末セルロース（アビセルを含む）、古紙類、瀘紙、一般紙類、木材、麦わら、稲わら、ふすま、もみがら、およびバガス、窒素源として、硫安、硝安などの無機アンモニウム塩、尿素、アミノ酸、肉エキス、酵母エキス、ポリペプトン、およびタンパク質分解物などの有機窒素含有物、ならびに無機塩類として、硫酸マグネシウム、リン酸２水素カリウム、酒石酸カリウム、硫酸亜鉛、硫酸マグネシウム、硫酸銅、塩化カルシウム、塩化鉄、塩化マンガン等を含むことができる。必要ならば有機微量栄養物を含有する培地を使用してもよい。培地は、寒天やゼラチンを加えて固化した固体培地、低濃度の寒天を加えた半流動培地、培地成分のみを入れた液体培地（ブイヨン、またはブロスともいう）を用いることができるが、液体培地が好ましい。 Medium is inorganic powder such as powdered cellulose (including Avicel), waste paper, straw paper, general paper, wood, straw, rice straw, bran, rice bran, bagasse, nitrogen source as ammonium source Organic nitrogen-containing materials such as ammonium salts, urea, amino acids, meat extracts, yeast extracts, polypeptone, and protein degradation products, and inorganic salts include magnesium sulfate, potassium dihydrogen phosphate, potassium tartrate, zinc sulfate, magnesium sulfate, sulfuric acid Copper, calcium chloride, iron chloride, manganese chloride and the like can be included. If necessary, a medium containing organic micronutrients may be used. The medium can be a solid medium solidified by adding agar or gelatin, a semi-fluid medium added with low-concentration agar, or a liquid medium (also referred to as bouillon or broth) containing only medium components. Is preferred.
本発明において、炭素源として培地に加える粉末セルロールおよびラクトースの量は、それぞれ、１０〜１００ｇ／Ｌおよび１０〜５０ｇ／Ｌ、好ましくは、４０〜５０ｇ／Ｌおよび５〜２０ｇ／Ｌである。粉末セルロールおよびラクトースの量が、それぞれ５０ｇ／Ｌおよび１０ｇ／Ｌ、あるいはそれぞれ４０ｇ／Ｌおよび２０ｇ／Ｌである場合に好ましい生産量が得られる。培養温度および培養時間は、セルラーゼ生産菌の種類によって異なるが、通常、２８〜３２℃、４８時間〜１０日ほど培養を行う。 In the present invention, the amounts of powdered cellulose and lactose added to the medium as a carbon source are 10 to 100 g / L and 10 to 50 g / L, preferably 40 to 50 g / L and 5 to 20 g / L, respectively. Preferred yields are obtained when the amounts of powdered cellulose and lactose are 50 g / L and 10 g / L, respectively, or 40 g / L and 20 g / L, respectively. The culture temperature and the culture time vary depending on the type of cellulase-producing bacteria, but are usually cultured at 28 to 32 ° C. for 48 hours to 10 days.
培養に用いることができる発酵槽としては、通気撹拌型、気泡塔型、流動層型、充填層型などが挙げられる。 Examples of the fermenter that can be used for the culture include an aeration stirring type, a bubble column type, a fluidized bed type, and a packed bed type.
上記培養液から、遠心分離、濾過などの公知の方法によって菌体を除去し上清液を得る。この上清液は、このまま粗酵素液として使用することが可能である。 The cells are removed from the culture solution by a known method such as centrifugation or filtration to obtain a supernatant. This supernatant can be used as a crude enzyme solution as it is.
（セルラーゼの精製）
セルラーゼは、上記上清液より、タンパク質精製に用いられる公知の方法、例えば、硫安塩析、有機溶媒（エタノール、メタノール、アセトン等）による沈殿分離、イオン交換クロマトグラフィー、等電点クロマトグラフィー、ゲルろ過クロマトグラフィー、疎水性クロマトグラフィー、吸着カラムクロマトグラフィー、基質または抗体などを利用したアフィニティークロマトグラフィー、逆相カラムクロマトグラフィーなどのクロマトグラフィー、精密ろ過、限外ろ過、逆浸透ろ過等の濾過処理など、を1つまたは複数組み合わせて用いて精製することが可能である。
(Purification of cellulase)
Cellulase is a known method used for protein purification from the above supernatant, for example, ammonium sulfate salting out, precipitation separation with an organic solvent (ethanol, methanol, acetone, etc.), ion exchange chromatography, isoelectric point chromatography, gel Filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using substrates or antibodies, chromatography such as reverse phase column chromatography, filtration treatment such as microfiltration, ultrafiltration, reverse osmosis filtration, etc. , Can be purified using one or a combination of two or more.
（セルラーゼの固定化）
精製したセルラーゼを、固定化して用いることもできる。固定化することによって、安定化され、連続反復使用が可能となる点において有効である。セルラーゼの固定化は、担体結合法、架橋法、包括法を用いて行うことができる。担体結合法では、セルラーゼを水不溶性の担体（例えば、ポリアクリルアミドゲル、ポリスチレン樹脂、多孔性ガラス、金属酸化物など）に、物理的吸着、イオン結合および共有結合を用いて結合させることができる。架橋法では、２個またはそれ以上の官能基を持つ試薬を用いて、セルラーゼとセルラーゼを架橋することによって固定化する。架橋試薬としては、Schiff塩基をつくるグルタルアルデヒド、ペプチド結合をするイソシアン酸誘導体、N,N’-エチレンマレイミド、ジアゾカップリングをするビスジアゾベンジン、あるいはアルキル化するN,N’-ポリメチレンビスヨードアセトアミドなどを用いることができる。包括法では、高分子ゲルの細かい格子の中にセルラーゼを取り込む格子型と、半透膜の高分子の皮膜によってセルラーゼを皮膜するマイクロカプセル型を用いる。格子型の方法では、合成高分子物質のポリアクリルアミドゲル、ポリビニルアルコール、光硬化性樹脂などの高分子化合物を用いることができる。マイクロカプセル型の方法では、ヘキサメチレンジアミン、セバコイルクロリド、ポリスチレン、レシチンなどを用いることができる（福井三郎、千畑一郎、鈴木周一、「酵素工学」東京化学同人発行、１９８１年）。
(Immobilization of cellulase)
Purified cellulase can be immobilized and used. Immobilization is effective in that it can be stabilized and used repeatedly continuously. Cellulase can be immobilized using a carrier binding method, a crosslinking method, or a comprehensive method. In the carrier binding method, cellulase can be bound to a water-insoluble carrier (eg, polyacrylamide gel, polystyrene resin, porous glass, metal oxide, etc.) using physical adsorption, ionic bonding, and covalent bonding. In the crosslinking method, cellulase and cellulase are immobilized by crosslinking using a reagent having two or more functional groups. Cross-linking reagents include glutaraldehyde that forms Schiff bases, isocyanic acid derivatives that form peptide bonds, N, N'-ethylenemaleimide, bisdiazobenzines that perform diazo coupling, or N, N'-polymethylene bisiodo that performs alkylation. Acetamide or the like can be used. The inclusion method uses a lattice type in which cellulase is incorporated into a fine lattice of a polymer gel and a microcapsule type in which cellulase is coated with a semipermeable polymer film. In the lattice-type method, a high molecular compound such as a polyacrylamide gel, polyvinyl alcohol, or a photocurable resin, which is a synthetic polymer substance, can be used. In the microcapsule type method, hexamethylenediamine, sebacoyl chloride, polystyrene, lecithin and the like can be used (Saburo Fukui, Ichiro Chibata, Shuichi Suzuki, “Enzyme Engineering”, published by Tokyo Chemical Dojin, 1981).
（セルラーゼ活性の測定）
セルラーゼ活性は、上記上清液または精製したセルラーゼに、濾紙、カルボキシメチルセルロース（CMC）、微結晶セルロース（Ａｖｉｃｅｌ）、サリシンおよびセロビオースなどの基質を加えて、一定時間酵素反応を行わせた後に、生じた還元糖をSomogy-Nelson法およびＤＮＳ法などにより発色させ所定の波長で比色定量して測定することが可能である。
(Measurement of cellulase activity)
Cellulase activity occurs after adding a substrate such as filter paper, carboxymethyl cellulose (CMC), microcrystalline cellulose (Avicel), salicin and cellobiose to the supernatant or purified cellulase and allowing the enzyme reaction to proceed for a certain period of time. It is possible to measure the color of the reduced sugar by colorimetric determination at a predetermined wavelength by coloring with the Somogy-Nelson method and DNS method.
Somogy-Nelson法においては、一定時間反応させた上記反応溶液にSomogy銅試薬（和光純薬）を加えて反応を停止する。その後およそ２０分間煮沸し、煮沸終了後急速に水道水にて冷却する。冷却後、Nelson試薬を注入して還元銅沈殿を溶解し発色させ、およそ３０分静置した後蒸留水を加え、吸光度を測定する。 In the Somogy-Nelson method, the reaction is stopped by adding a Somogy copper reagent (Wako Pure Chemical Industries, Ltd.) to the reaction solution reacted for a certain period of time. Then boil for about 20 minutes, and rapidly cool with tap water after boiling. After cooling, the Nelson reagent is injected to dissolve the reduced copper precipitate to develop color, and after standing for about 30 minutes, distilled water is added and the absorbance is measured.
ＤＮＳ法を用いる場合は、１％ CMC基質液に酵素液を加え、一定時間酵素反応を行わせたのち、煮沸などによって酵素反応を停止する。この反応液にジニトロサリチル酸を加えて、5分間煮沸し、冷却後吸光度を測定する（柏木豊「発酵糸状菌の酵素」、微生物遺伝資源利用マニュアル（１６）、独立行政法人農業生物資源研究所発行、2004年2月29日発行）。 When using the DNS method, an enzyme solution is added to a 1% CMC substrate solution, the enzyme reaction is performed for a certain period of time, and then the enzyme reaction is stopped by boiling or the like. Dinitrosalicylic acid is added to this reaction solution, boiled for 5 minutes, and after cooling, the absorbance is measured (Yutaki Yutaka “Enzyme of Fermented Filamentous Fungi”, Microbial Genetic Resource Use Manual (16), published by National Institute of Agrobiological Sciences) Published February 29, 2004).
（セルロースの分解または糖化）
セルロースの分解または糖化は、公知の方法を用いることができる。例えば、セルロース系物質を水性媒体中に懸濁し、セルラーゼを含む培養上清または精製したセルラーゼを加え、撹拌または振とうしながら加温して、セルロース系物質を分解または糖化することができる。この方法において、反応液のｐＨおよび温度は、セルラーゼが失活しない範囲内であればよく、一般的に、常圧で反応を行う場合、温度は５〜９５℃、ｐＨは１〜１１の範囲でよい。例えば、５０ｇの粉砕した稲わらに、０．２５〜１Lの酢酸緩衝液（０．０５M、pH ４．８）および０．０１〜０．２Lのセルラーゼを含む培養上清または精製したセルラーゼを添加して、４５〜６０℃で攪拌または振とうしながら、セルロースの分解または糖化を行う。また、この酵素反応は、バッチ式で行っても、連続式で行ってもよい。
(Decomposition or saccharification of cellulose)
A known method can be used for the decomposition or saccharification of cellulose. For example, a cellulosic material can be suspended or suspended in an aqueous medium, a culture supernatant containing cellulase or purified cellulase can be added, and heated with stirring or shaking to decompose or saccharify the cellulosic material. In this method, the pH and temperature of the reaction solution may be within the range where cellulase is not inactivated. Generally, when the reaction is carried out at normal pressure, the temperature is in the range of 5 to 95 ° C. and the pH is in the range of 1 to 11. It's okay. For example, to 50 g of ground rice straw, a culture supernatant or purified cellulase containing 0.25 to 1 L of acetate buffer (0.05 M, pH 4.8) and 0.01 to 0.2 L of cellulase is added. Then, the cellulose is decomposed or saccharified while stirring or shaking at 45 to 60 ° C. Moreover, this enzyme reaction may be performed by a batch type or a continuous type.
次に実施例を挙げ、本発明を更に詳しく説明するが、本発明はこれらに限定されるものではない。
（セルラーゼ生産菌の培養）
本実施例においては、セルラーゼ生産菌としてアクレモニウム・セルロリティカス（Acremonium cellulolyticus）Ｃ１株（ＦＥＲＭ Ｐ−１８５０８）を用いた。培地は下記の組成を有するセルラーゼ生産菌培養培地を用いた。
EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not limited to these.
(Culture of cellulase-producing bacteria)
In this example, Acremonium cellulolyticus C1 strain (FERM P-18508) was used as a cellulase-producing bacterium. As the medium, a cellulase-producing bacterial culture medium having the following composition was used.
〔培地の組成〕：
炭素源 ５０ｇ／Ｌまたは ６０ｇ／Ｌ
硫酸アンモニウム ５ｇ／Ｌ
尿素 ４ｇ／Ｌ
硫酸マグネシウム １．２ｇ／Ｌ
リン酸２水素カリウム ２４ｇ／Ｌ
酒石酸カリウム ４．７ｇ／Ｌ
硫酸亜鉛 １０ ｍｇ／Ｌ
硫酸マンガン １０ ｍｇ／Ｌ
硫酸銅 １０ ｍｇ／Ｌ
Ｔｗｅｅｎ ８０ １ ｇ／Ｌ
ｐＨ ４．０
[Composition of medium]:
Carbon source 50 g / L or 60 g / L
Ammonium sulfate 5g / L
Urea 4g / L
Magnesium sulfate 1.2g / L
Potassium dihydrogen phosphate 24g / L
Potassium tartrate 4.7g / L
Zinc sulfate 10 mg / L
Manganese sulfate 10 mg / L
Copper sulfate 10 mg / L
Tween 80 1 g / L
pH 4.0
上記炭素源としては、粉末セルロース４０ｇ／Ｌおよびトレハロース１０ｇ／Ｌ、粉末セルロース３０ｇ／Ｌおよびトレハロース２０ｇ／Ｌ、粉末セルロース４０ｇ／Ｌおよびセロビオース１０ｇ／Ｌ、粉末セルロース３０ｇ／Ｌおよびセロビオース２０ｇ／Ｌ、ラクトース５０ｇ／Ｌ、粉末セルロース４０ｇ／Ｌおよびラクトース１０ｇ／Ｌ、粉末セルロース５０ｇ／Ｌ、粉末セルロース５０ｇ／Ｌおよびラクトース１０ｇ／Ｌ、粉末セルロース４０ｇ／Ｌおよびラクトース２０ｇ／Ｌ、または粉末セルロース６０ｇ／Ｌを用いた。当該分野で公知である方法により殺菌した上記セルラーゼ生産菌培養培地にアクレモニウム・セルロリティカス（Acremonium cellulolyticus）Ｃ１株（ＦＥＲＭ Ｐ−１８５０８）を接種して３０℃で１０日間好気的に培養した。この培養液を遠心分離して得た上清液について、製造されたセルラーゼの酵素活性を測定した。 Examples of the carbon source include powdered cellulose 40 g / L and trehalose 10 g / L, powdered cellulose 30 g / L and trehalose 20 g / L, powdered cellulose 40 g / L and cellobiose 10 g / L, powdered cellulose 30 g / L and cellobiose 20 g / L, Lactose 50 g / L, powdered cellulose 40 g / L and lactose 10 g / L, powdered cellulose 50 g / L, powdered cellulose 50 g / L and lactose 10 g / L, powdered cellulose 40 g / L and lactose 20 g / L, or powdered cellulose 60 g / L Was used. The cellulase-producing bacterial culture medium sterilized by a method known in the art was inoculated with Acremonium cellulolyticus C1 strain (FERM P-18508) and aerobically cultured at 30 ° C. for 10 days. . With respect to the supernatant obtained by centrifuging this culture solution, the enzyme activity of the produced cellulase was measured.
（セルラーゼ活性の測定）
本実施例において、セルラーゼの酵素活性は、濾紙を基質として用いて行った。具体的には、濾紙（ワットマンＮｏ．１、１×６ｃｍ）を基質とし、これに適宜希釈した培養上清液０．５ｍＬとクエン酸緩衝液（ｐＨ４．８、０．０５Ｍ）１．０ｍＬを加え、５０℃で１．０時間酵素反応を行った後、ジニトロサリチル酸試薬３．０ｍＬを加え、１００℃で５分間加熱し発色させた。冷却後、蒸留水２．５ｍＬにこれを２００μｌ加え、５４０ｎｍの波長で比色定量した。
(Measurement of cellulase activity)
In this example, the enzyme activity of cellulase was performed using filter paper as a substrate. Specifically, filter paper (Whatman No. 1, 1 × 6 cm) is used as a substrate, and 0.5 mL of culture supernatant diluted appropriately and 1.0 mL of citrate buffer (pH 4.8, 0.05 M) are added thereto. In addition, after carrying out an enzyme reaction at 50 ° C. for 1.0 hour, 3.0 mL of a dinitrosalicylic acid reagent was added, and the color was developed by heating at 100 ° C. for 5 minutes. After cooling, 200 μl of this was added to 2.5 mL of distilled water and colorimetrically determined at a wavelength of 540 nm.
得られた結果を表１に示す。１分間に１μｍｏｌのグルコースに相当する還元糖を生成する酵素量を１ユニット（Ｕ）として示す。 The obtained results are shown in Table 1. The amount of enzyme that produces a reducing sugar corresponding to 1 μmol of glucose per minute is shown as 1 unit (U).
表１から明らかなように、粉末セルロース（５０ｇ／Ｌ）のみを培地に添加した培養液に由来する上清液の酵素活性は、１１．７Ｕ／ｍＬであるのに対して、粉末セルロースとトレハロースまたは粉末セルロースとセロビオースを培地に添加した培養液に由来する上清液の酵素活性は、それぞれ、８．８もしくは９．１Ｕ／ｍＬまたは９．７もしくは９．４Ｕ／ｍＬであり、やや低かった。一方、粉末セルロースとラクトース（それぞれ、４０ｇ／Ｌと１０ｇ／Ｌ）を培地に添加した培養液に由来する上清液では、粉末セルロースとトレハロースまたはセロビオースを添加した場合と比べて高い酵素活性を示した（１３．９Ｕ／ｍＬ）。また、ラクトース（５０ｇ／Ｌ）を唯一の炭素源として培地に添加した場合、上清液の酵素活性が大幅に低くなることが観察された（３．９Ｕ／ｍＬ）。さらに、６０ｇ／Ｌの粉末セルロースのみを炭素源として培養したときの上清液中の酵素活性は１３．２Ｕ／ｍＬであるのに対して、５０ｇ／Ｌの粉末セルロースと１０ｇ／Ｌのラクトースまたは４０ｇ／Ｌの粉末セルロースと２０ｇ／Ｌのラクトースを培地に添加した培養液に由来する上清液中の酵素活性は、それぞれ１９．５または１９．０Ｕ／ｍＬであり、約１．５倍に増大していた。 As is apparent from Table 1, the enzymatic activity of the supernatant derived from the culture solution in which only powdered cellulose (50 g / L) was added to the medium was 11.7 U / mL, whereas powdered cellulose and trehalose Alternatively, the enzyme activities of the supernatants derived from the culture solution in which powdered cellulose and cellobiose were added to the medium were 8.8 or 9.1 U / mL or 9.7 or 9.4 U / mL, respectively, which were slightly low . On the other hand, the supernatant derived from the culture solution in which powdered cellulose and lactose (40 g / L and 10 g / L, respectively) were added to the medium showed higher enzyme activity than when powdered cellulose and trehalose or cellobiose were added. (13.9 U / mL). In addition, when lactose (50 g / L) was added to the medium as the sole carbon source, it was observed that the enzyme activity of the supernatant was significantly reduced (3.9 U / mL). Furthermore, the enzyme activity in the supernatant when culturing only 60 g / L of powdered cellulose as a carbon source is 13.2 U / mL, whereas 50 g / L of powdered cellulose and 10 g / L of lactose or Enzyme activity in the supernatant liquid derived from the culture solution in which 40 g / L of powdered cellulose and 20 g / L of lactose were added to the medium was 19.5 or 19.0 U / mL, respectively, about 1.5 times It was increasing.
これらの結果より、粉末セルロースとラクトースの両方を培地に添加した場合に、セルラーゼの生産量が増大することが明らかとなった。 From these results, it was revealed that the production amount of cellulase increases when both powdered cellulose and lactose are added to the medium.
本発明は、粉末セルロースとラクトースとを添加した培地中にてセルラーゼ生産菌を培養することによってセルラーゼの生産量を高めることができるために、セルラーゼを効率的に製造できるとともに、セルロースの分解、糖化も、安価、効率的に行いうる点で有用である。 The present invention can increase the amount of cellulase produced by culturing cellulase-producing bacteria in a medium supplemented with powdered cellulose and lactose, so that cellulase can be produced efficiently and cellulose is decomposed and saccharified. Is also useful in that it can be performed inexpensively and efficiently.
Claims (9)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2007118021A JP4998991B2 (en) | 2007-04-27 | 2007-04-27 | Method for producing cellulase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2007118021A JP4998991B2 (en) | 2007-04-27 | 2007-04-27 | Method for producing cellulase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2008271826A true JP2008271826A (en) | 2008-11-13 |
| JP4998991B2 JP4998991B2 (en) | 2012-08-15 |
Family
ID=40050607
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2007118021A Active JP4998991B2 (en) | 2007-04-27 | 2007-04-27 | Method for producing cellulase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4998991B2 (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503865B (en) * | 2009-03-23 | 2010-08-25 | 江南大学 | Method for preparing mlcrocrystalline cellulose by corps straw |
| CN102002875A (en) * | 2010-10-23 | 2011-04-06 | 吉林大学 | Method for preparing microcrystalline cellulose by using carrot pomace |
| JP2012105568A (en) * | 2010-11-16 | 2012-06-07 | Shinshu Univ | Method for impasting food material containing mannan, and impasting agent for food material containing mannan |
| WO2013190875A1 (en) * | 2012-06-21 | 2013-12-27 | 月島機械株式会社 | Biomass processing system and processing method |
| CN104963228A (en) * | 2015-07-16 | 2015-10-07 | 吉林大学 | Method for preparing nano cellulose by utilizing sunflower seed husks |
| CN107190546A (en) * | 2017-06-06 | 2017-09-22 | 湖南工业大学 | A kind of method that utilization oil-tea camellia husks prepare the nanofibrils cellulose of high length-diameter ratio |
| WO2019059404A1 (en) | 2017-09-25 | 2019-03-28 | 味の素株式会社 | Protein production method and disaccharide production method |
| WO2019073954A1 (en) | 2017-10-10 | 2019-04-18 | 味の素株式会社 | Method for manufacturing protein |
| WO2021251383A1 (en) | 2020-06-11 | 2021-12-16 | 味の素株式会社 | Method for producing protein |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003135052A (en) * | 2001-11-02 | 2003-05-13 | National Institute Of Advanced Industrial & Technology | Method for hydrolyzing cellulose raw material |
-
2007
- 2007-04-27 JP JP2007118021A patent/JP4998991B2/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003135052A (en) * | 2001-11-02 | 2003-05-13 | National Institute Of Advanced Industrial & Technology | Method for hydrolyzing cellulose raw material |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503865B (en) * | 2009-03-23 | 2010-08-25 | 江南大学 | Method for preparing mlcrocrystalline cellulose by corps straw |
| CN102002875A (en) * | 2010-10-23 | 2011-04-06 | 吉林大学 | Method for preparing microcrystalline cellulose by using carrot pomace |
| JP2012105568A (en) * | 2010-11-16 | 2012-06-07 | Shinshu Univ | Method for impasting food material containing mannan, and impasting agent for food material containing mannan |
| WO2013190875A1 (en) * | 2012-06-21 | 2013-12-27 | 月島機械株式会社 | Biomass processing system and processing method |
| JP2014003913A (en) * | 2012-06-21 | 2014-01-16 | Tsukishima Kikai Co Ltd | Treatment system and treatment method for biomass |
| CN104963228A (en) * | 2015-07-16 | 2015-10-07 | 吉林大学 | Method for preparing nano cellulose by utilizing sunflower seed husks |
| CN107190546A (en) * | 2017-06-06 | 2017-09-22 | 湖南工业大学 | A kind of method that utilization oil-tea camellia husks prepare the nanofibrils cellulose of high length-diameter ratio |
| WO2019059404A1 (en) | 2017-09-25 | 2019-03-28 | 味の素株式会社 | Protein production method and disaccharide production method |
| WO2019073954A1 (en) | 2017-10-10 | 2019-04-18 | 味の素株式会社 | Method for manufacturing protein |
| WO2021251383A1 (en) | 2020-06-11 | 2021-12-16 | 味の素株式会社 | Method for producing protein |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4998991B2 (en) | 2012-08-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4986038B2 (en) | Method for producing highly hydrolyzed cellulase and hemicellulase | |
| JP4998991B2 (en) | Method for producing cellulase | |
| JP6929499B2 (en) | Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzyme | |
| WO2000039288A1 (en) | Polypeptides | |
| CA2650810A1 (en) | Thermostable cellulase and methods of use | |
| JP2019503688A (en) | Lactic acid-utilizing bacteria genetically modified to secrete polysaccharide degrading enzymes | |
| CN112063605B (en) | Method for preparing gentiooligosaccharide by catalyzing cellulose with complex enzyme and application of method | |
| Hetzler et al. | Saccharification of cellulose by recombinant Rhodococcus opacus PD630 strains | |
| Zhao et al. | Expression and characterization of GH3 β-Glucosidase from Aspergillus niger NL-1 with high specific activity, glucose inhibition and solvent tolerance | |
| CN104046586B (en) | One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof | |
| Mól et al. | β-glucosidase: An overview on immobilization and some aspects of structure, function, applications and cost | |
| CN108291245A (en) | Use high concentration sugar mixture inducible gene expression | |
| CN109456954B (en) | Beta-glucosidase mutant with improved thermal stability and application thereof | |
| JP6103584B2 (en) | Filamentous fungal mutants in which the cre1 gene does not function | |
| JP6518107B2 (en) | Transcription factor mutant | |
| Ryabtseva et al. | Biotechnology of lactulose production: progress, challenges, and prospects | |
| CN101824401B (en) | Glucanase and coding nucleic acid and expression thereof | |
| JP2785323B2 (en) | β-glucosidase and method for producing the same | |
| CN109504669B (en) | Beta-glucosidase with improved resistance to trypsin | |
| KR101236558B1 (en) | A novel isolated Phoma sp. from rotten tangerine peel and cellulolytic enzymes and β-glucosidase produced by thereof | |
| Adelabu | The Cellulase production by immobilized cells of Candida tropicalis isolated from grasshopper Zonocerus variegatus in Saw dust and rice husk medium: Production of cellulase enzyme by immobilized yeast cell | |
| Karthikeyan et al. | Purification and Characterization of Cellulase from Aspergillus niger | |
| Long et al. | Patents and literature | |
| CN116064700A (en) | Sophorose, and preparation method and application thereof | |
| CN118086256A (en) | Medium-temperature alpha-amylase and gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20091112 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20091109 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20120124 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20120326 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20120417 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20120510 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 Ref document number: 4998991 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20150525 Year of fee payment: 3 |
|
| S531 | Written request for registration of change of domicile |
Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313115 |
|
| S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |