CN114958623A - 一株高产纤维素酶的盖姆斯木霉及其应用 - Google Patents
一株高产纤维素酶的盖姆斯木霉及其应用 Download PDFInfo
- Publication number
- CN114958623A CN114958623A CN202210718406.8A CN202210718406A CN114958623A CN 114958623 A CN114958623 A CN 114958623A CN 202210718406 A CN202210718406 A CN 202210718406A CN 114958623 A CN114958623 A CN 114958623A
- Authority
- CN
- China
- Prior art keywords
- trichoderma
- culture medium
- culture
- strain
- cellulase
- 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 32
- 229940106157 cellulase Drugs 0.000 title claims abstract description 29
- 241000944293 Trichoderma gamsii Species 0.000 title claims description 41
- 238000004519 manufacturing process Methods 0.000 title claims description 14
- 239000010902 straw Substances 0.000 claims abstract description 49
- 239000007787 solid Substances 0.000 claims abstract description 34
- 238000000855 fermentation Methods 0.000 claims abstract description 21
- 230000004151 fermentation Effects 0.000 claims abstract description 21
- 239000000758 substrate Substances 0.000 claims abstract description 17
- 240000008042 Zea mays Species 0.000 claims abstract description 15
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 15
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 15
- 235000005822 corn Nutrition 0.000 claims abstract description 15
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 14
- 241000209140 Triticum Species 0.000 claims abstract description 14
- 235000021307 Triticum Nutrition 0.000 claims abstract description 14
- 235000009566 rice Nutrition 0.000 claims abstract description 14
- 241000223259 Trichoderma Species 0.000 claims abstract description 13
- 241000196252 Ulva Species 0.000 claims abstract description 10
- 240000004731 Acer pseudoplatanus Species 0.000 claims abstract description 9
- 235000002754 Acer pseudoplatanus Nutrition 0.000 claims abstract description 9
- 235000006485 Platanus occidentalis Nutrition 0.000 claims abstract description 9
- 108010047754 beta-Glucosidase Proteins 0.000 claims abstract description 7
- 102000006995 beta-Glucosidase Human genes 0.000 claims abstract description 7
- 240000007594 Oryza sativa Species 0.000 claims abstract 3
- 239000000843 powder Substances 0.000 claims description 46
- 239000001963 growth medium Substances 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 18
- 241000234642 Festuca Species 0.000 claims description 12
- 239000001913 cellulose Substances 0.000 claims description 10
- 229920002678 cellulose Polymers 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 8
- 238000009629 microbiological culture Methods 0.000 claims description 7
- 239000011159 matrix material Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 241000196253 Ulva prolifera Species 0.000 claims description 4
- 230000000593 degrading effect Effects 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 101710112457 Exoglucanase Proteins 0.000 claims description 2
- 230000000050 nutritive effect Effects 0.000 claims 2
- 238000009630 liquid culture Methods 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 57
- 108090000790 Enzymes Proteins 0.000 abstract description 57
- 229940088598 enzyme Drugs 0.000 abstract description 57
- 230000000694 effects Effects 0.000 abstract description 33
- 230000006698 induction Effects 0.000 abstract description 20
- 241000227728 Trichoderma hamatum Species 0.000 abstract description 17
- 239000007788 liquid Substances 0.000 abstract description 13
- 230000015556 catabolic process Effects 0.000 abstract description 6
- 238000006731 degradation reaction Methods 0.000 abstract description 6
- 101100256850 Drosophila melanogaster EndoA gene Proteins 0.000 abstract description 3
- 240000007171 Imperata cylindrica Species 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 19
- 239000000243 solution Substances 0.000 description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 241000209094 Oryza Species 0.000 description 11
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 235000010980 cellulose Nutrition 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 8
- 241000355691 Trichoderma reesei QM9414 Species 0.000 description 7
- IFBHRQDFSNCLOZ-YBXAARCKSA-N 4-nitrophenyl-beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CC=C([N+]([O-])=O)C=C1 IFBHRQDFSNCLOZ-YBXAARCKSA-N 0.000 description 6
- 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 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000010563 solid-state fermentation Methods 0.000 description 6
- 244000005700 microbiome Species 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 230000004580 weight loss Effects 0.000 description 5
- LWFUFLREGJMOIZ-UHFFFAOYSA-N 3,5-dinitrosalicylic acid Chemical compound OC(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1O LWFUFLREGJMOIZ-UHFFFAOYSA-N 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 244000025254 Cannabis sativa Species 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 101150074253 TEF1 gene Proteins 0.000 description 3
- 241001530102 Tabebuia Species 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 239000007979 citrate buffer Substances 0.000 description 3
- 229960004106 citric acid Drugs 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 239000002207 metabolite Substances 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 101100010928 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) tuf gene Proteins 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 2
- 229960002303 citric acid monohydrate Drugs 0.000 description 2
- 230000001332 colony forming effect Effects 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 240000004784 Cymbopogon citratus Species 0.000 description 1
- 235000017897 Cymbopogon citratus Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101000597041 Gallus gallus Transcriptional enhancer factor TEF-3 Proteins 0.000 description 1
- 101000653735 Homo sapiens Transcriptional enhancer factor TEF-1 Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102100020678 Krueppel-like factor 3 Human genes 0.000 description 1
- 101710116712 Krueppel-like factor 3 Proteins 0.000 description 1
- 241000208204 Linum Species 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 101100213970 Schizosaccharomyces pombe (strain 972 / ATCC 24843) ypt3 gene Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 101150001810 TEAD1 gene Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000012271 agricultural production Methods 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
- 238000003556 assay Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000002457 bidirectional effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 description 1
- 229940052299 calcium chloride dihydrate Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- ARPLKSKOWFTTTQ-UHFFFAOYSA-L cobalt(2+);dichloride;dihydrate Chemical compound O.O.Cl[Co]Cl ARPLKSKOWFTTTQ-UHFFFAOYSA-L 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000012263 liquid product Substances 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 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
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical class [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 235000011006 sodium potassium tartrate Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
- C08H8/00—Macromolecular compounds derived from lignocellulosic materials
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
- C12N9/2454—Dextranase (3.2.1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01011—Dextranase (3.2.1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
-
- 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
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Mycology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Polymers & Plastics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明公开了一株高产纤维素酶的木霉菌株及其应用。本发明菌株为盖姆斯木霉Trichoderma gamsii TC787菌株,其在液态摇瓶发酵条件下的滤纸酶活、内切酶活和β‑葡萄糖苷酶活分别达到2.21、0.41和1.45IU/mL,在固态发酵条件下,TC787在粉碎的水稻秸秆、玉米秸秆、浒苔、小麦秸秆、羊茅草和悬铃木叶片的诱导下,滤纸酶活分别为2.02、1.89、2.51、5.78、0.39和3.08IU/g干物质。该菌株对上述基物的降解率分别为21.9%、24.1%、57.0%、18.3%、13.0%和12.3%。盖姆斯木霉TC787菌株适用于液态发酵产纤维素酶和固态发酵降解木质纤维素。
Description
技术领域
本发明涉及真菌资源及其利用领域的一株高产纤维素酶的盖姆斯木霉及其应用。
背景技术
木质纤维素是地球上储量最大的可再生资源,其中纤维素是其主要成分,约占30%,有效降解纤维素使其转化为可利用的产品或实现农田秸秆分解是当前的研究热点。木霉属真菌是重要的纤维素降解菌,其分泌的纤维素酶能够将纤维素分解成还原糖。工业菌株酶活有待提高、市场成本高等问题阻碍了其应用,农业秸秆还田需要高酶活菌株。因此,筛选新的高产纤维素酶菌株具有重要的理论意义和应用前景。
发明内容
为解决生产中纤维素酶成本高和/或酶活低和/或农业秸秆还田需要高酶活菌株等问题,本发明提供一株高产纤维素酶的盖姆斯木霉菌株,以期高效分解纤维素,解决工农业生产中的问题。
为解决上述技术问题,本发明首先提供了一株盖姆斯木霉,所述盖姆斯木霉为盖姆斯木霉(Trichoderma gamsii),其菌株号为TC787,其在中国微生物菌种保藏管理委员会普通微生物中心的登记入册编号为CGMCC No.22416。以下简称盖姆斯木霉TC787。
本发明还提供了一种用于制备纤维素酶的培养物,所述培养物含有盖姆斯木霉TC787。
上述培养物中,所述培养物是将盖姆斯木霉TC787在微生物培养基中培养得到的物质(发酵产物)。
上述培养物中,所述物质包括所述盖姆斯木霉TC787和盖姆斯木霉TC787的代谢物。
上述培养物中,所述微生物培养基可为固体培养基或液体培养基。
术语“培养物”是指经人工接种和培养后,长有微生物群体的液体或固体产物(发酵产物)的统称。即通过将微生物进行生长和/或扩增而获得的产物,其可以是微生物的生物学纯培养物,也可以含有一定量的培养基、代谢物或培养过程中产生的其他成分。术语“培养物”还包括通过将微生物传代而获得的传代培养物,其可以是某一代的培养物,也可以是若干代的混合物。
上述盖姆斯木霉TC787或其培养物在制备纤维素酶中的应用也是本发明保护的范围。
本发明的另一个目的是提供一种制备纤维素酶的方法。
本发明所提供的制备纤维素酶的方法是利用盖姆斯木霉TC787进行发酵得到纤维素酶。
上述方法中,所述纤维素酶可为内切型葡聚糖酶、外切型葡聚糖酶和/或β-葡萄糖苷酶。
上述方法中,所述发酵包括用培养基培养所述盖姆斯木霉TC787,所述培养基可为液体培养基或固体培养基;所述固体培养基可含有基质粉,所述基质粉可来源于浒苔、水稻秸秆、玉米秸秆、小麦秸秆、羊茅草和悬铃木叶片中的至少一种。
上述方法中,所述固体培养基可由固体基物与基础营养盐培养基组成。
所述固体培养基中,所述固体基物与基础营养盐培养基的质量比可为1:2.4-2.6;1L所述基础营养盐培养基的组成可为:(NH4)2SO4 4.9-5.1g/L,KH2PO4 2.9-3.1g/L,MgSO4·7H2O 0.49-0.51g/L,CaCl2 0.49-0.51g/L,其余为水;所述固体基物由所述基质粉与麸皮组成,所述固体基物中,所述基质粉与所述麸皮的质量比可为1:1。
盖姆斯木霉TC787或其培养物在降解纤维素中的应用也是本发明保护的范围。
在上述降解纤维素中的应用中,所述纤维素可来源于下述至少一种:浒苔、水稻秸秆、玉米秸秆、小麦秸秆、羊茅草和悬铃木叶片。
实验证明,盖姆斯木霉TC787在液态发酵条件下,滤纸酶活、内切酶活和β-葡萄糖苷酶活力分别达到2.21、0.41和1.45IU/mL发酵液,分别是相同条件下里氏木霉QM9414酶活的2.22倍、0.85倍和3.30倍。表明盖姆斯木霉TC787在实验室条件下液态发酵具有良好的产纤维素酶能力。在固态发酵条件下,盖姆斯木霉TC787在水稻秸秆、玉米秸秆、浒苔、小麦秸秆、羊茅草和悬铃木叶片的诱导下,均能够诱导产纤维素酶,发酵6天,滤纸酶活分别为2.02、1.89、2.51、5.78、0.39和3.08IU/g干物质(如图3所示)。盖姆斯木霉TC787对上述6种基物的降解率(失重率)分别为21.9%、24.1%、57.0%、18.3%、13.0%和12.3%(如图4所示)。在固态发酵条件下,盖姆斯木霉TC787表现出良好的基物降解能力和产纤维素酶性能。盖姆斯木霉TC787液态和固态发酵均展现出良好的产纤维素酶潜力,是一株有应用潜力的真菌资源,其产酶性能良好,有工业生产纤维素酶和农作物秸秆降解还田循环利用的优势。
保藏说明
菌种名称:盖姆斯木霉
拉丁名:Trichoderma gamsii
菌株编号:TC787
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称:CGMCC
地址:北京市朝阳区北辰西路1号院3号
保藏日期:2021年04月23日
保藏中心登记入册编号:CGMCC No.22416
附图说明
图1为盖姆斯木霉TC787的形态特征。
a-c.25℃培养7天的菌落形态(a.CMD,b.PDA,c.SNA);d,e.产孢簇(d.CMD,e.SNA);f-l.分生孢子梗和瓶梗;m.厚垣孢子;n,o.分生孢子。标尺:a–c=20mm;d,e=200μm;f-h=20μm;i,j=10μm;k-o=5μm。
图2为液态发酵条件下盖姆斯木霉TC787与里氏木霉QM9414发酵192h产酶能力的对比。
图3为固态发酵条件下盖姆斯木霉TC787对6种植物性基物的降解能力。
图4为固态发酵条件下6种不同基物对盖姆斯木霉TC787产纤维素酶的诱导。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例中的定量实验,如无特别说明,实验均设置三次重复。
实施例1、盖姆斯木霉TC787 CGMCC No.22416的分离、纯化、保藏和鉴定
1.盖姆斯木霉TC787 CGMCC No.22416的分离纯化和保存
土壤样品取自黑龙江省大兴安岭南瓮河自然保护区,称1g土壤样品,用无菌水稀释至10-1、10-2和10-3三个浓度,每个浓度取200μl涂布于PDA培养基(马铃薯200g,葡萄糖20g,琼脂20g,蒸馏水定容至1000mL,高压灭菌,添加氯霉素300mg/L)平板表面。在25℃下将平板倒置培养1–3天后,挑取单菌落转移至PDA斜面培养基保存。取编号为TC787的菌株(即盖姆斯木霉TC787)进行下述鉴定。
2.盖姆斯木霉TC787的鉴定
盖姆斯木霉TC787生物学特性(如图1所示):在CMD培养基(玉米粉40g/L,葡萄糖20g/L,琼脂20g/L,其余为水)上,25℃培养3天后菌落半径65-67mm,3天长满平板;菌落近无色,放射状,轮廓清晰,气生菌丝丰富,集中在菌落边缘,羊毛状,延伸到培养皿盖;分生孢子15天后产生于产孢簇内,产孢簇位于气生菌丝上,数量较多,白色,直径<1mm;厚垣孢子常见。在PDA培养基上,25℃培养3天后菌落直径69-70mm,3天长满平板,菌落与CMD培养基上的类似,但气生菌丝更丰富;分生孢子5天后产生于气生菌丝上;厚垣孢子丰富。在SNA培养基(KH2PO4 1g/L,KNO31g/L,MgSO4·7H2O 0.5g/L,KCl 0.5g/L,葡萄糖0.2g/L,蔗糖0.2g/L,琼脂20g/L,其余为水)上,25℃培养3天后菌落半径41-46mm,5天长满平板,菌落与CMD培养基上的类似;分生孢子5天后产生于气生菌丝上,7天后出现产孢簇,产孢簇较少,主要分布在菌落边缘,初白色,8天后变为绿色;分生孢子梗trichoderma型;瓶梗单生,对生或3轮生,窄烧瓶型,少数锥形,8.1-12.4(-14.2)×2.1-3.1μm,长宽比2.9-5.0(-6.0),基部宽1.4-2.5μm;分生孢子绿色,形状多变,球形,近球形,卵圆形或椭球形,表面光滑,具若干滴状斑点,3.3-4.7(-5.6)×2.8-3.6μm,长宽比1.0-1.5;厚垣孢子常见,顶生。该菌株在上述三种培养基上均无明显气味和色素产生。
基于盖姆斯木霉TC787的两个DNA片段(ITS、tef1)的分子生物学鉴定:
(1)采用CTAB法提取真菌基因组;
(2)ITS和tef1基因片段的扩增及测序:
PCR扩增引物和测序引物分别见表1和表2,PCR反应体系和反应程序分别见表3和表4。反应结束后取2μL扩增产物进行1.0%琼脂糖凝胶电泳检测,凝胶成像系统拍照,由测序公司进行双向测序,获得碱基序列。
表1 PCR扩增引物
表2 PCR测序引物
表3 PCR反应体系(25μL)
表4 PCR反应程序
(3)序列分析
利用Clustal X程序和Bioedit等软件对测序结果进编辑和分析。
用引物ITS4、ITS5测序拼接后得到的ITS序列见SEQ ID No.1。
用引物TEF-1和TEF-2测序拼接后得到的tef1序列见SEQ ID No.2。
将获得的序列与GenBank上的进行比对。结合形态学特征、培养特性和DNA序列数据,将TC787菌株鉴定为盖姆斯木霉(Trichoderma gamsii)。
3.盖姆斯木霉TC787菌株的保藏
盖姆斯木霉TC787已于2021年4月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.22416。
实施例2、盖姆斯木霉TC787和里氏木霉QM9414液体发酵产酶特性研究
1.液体发酵
1)菌株活化:将斜面保存的盖姆斯木霉TC787和里氏木霉QM9414(TRICHODERMAREESEI QM 9414)(Lars G.FAGERSTAM and L.G&an PETTERSSON.THE CELLULOLYTICCOMPLEX OF TRICHODERMA REESEI QM 9414.Volume 98,number 2.FEBSLETTERS.February 1979)在PDA平板上活化,25℃培养3–5d,待其产生大量的分生孢子。向平板中加入5–10mL无菌水,使用1000μL移液枪轻吸打匀。为了避免培养基中残留物的影响,将收集的分生孢子液通过200目过滤筛过滤,收集滤液,4℃,2000g离心,去除上清,备用。吸取少量悬浮液进行稀释(根据悬浮液浓度,稀释10-103倍不等),利用血球计数板进行计数。
2)孢子接种培养:调节孢子悬浮液浓度为5×106cfu/mL(每毫升中的菌落形成单位,即孢子数目),接种至100mL酶诱导培养基中,25℃,180r/min,恒温培养192h,每个处理设置3次重复。其中,酶诱导培养基(1L):微晶纤维素粉10.0g,Protease peptone 1.0g,磷酸氢二钾2.0g,硫酸铵1.4g,七水硫酸镁0.3g,二水氯化钙0.3g,尿素0.3g,七水硫酸亚铁5.0mg,一水硫酸锰1.6mg,七水硫酸锌1.4mg,二水氯化钴2.0mg,加蒸馏水定容,调pH至5.0,115℃,20min高压蒸汽灭菌。待发酵结束后,取2mL发酵液经过12000r/min离心15min后,立即取上清液进行酶活检测。上述实验重复3次,每次重复采用100ml PD培养基(马铃薯200g,葡萄糖20g,蒸馏水定容至1000mL,高压灭菌得到的液体培养基)进行发酵。
2、木质纤维素酶活性测定
1)滤纸酶酶活的测定方法(DNS法):在容积为1.5mL的离心管中,置入50μL pH4.8,50mMol/L(以下简称mM)的柠檬酸缓冲液(一水柠檬酸210g,无菌水750mL,加入NaOH(50-60g),稀释至1000ml,获得pH 4.5的1mol/L柠檬酸缓冲液。当稀释至50mM时,pH值为4.8)和1cm×1cm的Whatman No.1滤纸条。加入适当稀释的酶液25μL,50℃恒温水浴1h。加入150μL DNS溶液(Ghose,1987,Distilled water 1416.0mL,3,5-Dinitrosalicylic acid10.6g,NaOH 19.8g,溶解后加入:Rochelle salts 306.0g,Phenol(melt at 50℃)7.6mL,Na2S2O5 8.3g,配置完成后,棕色瓶保存,静置7天后备用),沸水浴10min,冷水冷却。加入1mLddH2O,OD540 nm下测定光吸收值,测算酶活力。每个处理设置3次重复,灭活酶液作为对照。滤纸酶酶活力按照国际单位定义为:在pH 4.8,50℃下每分钟催化水解生成1μmol葡萄糖的酶量为一个酶活力单位IU。
2)内切酶酶活(羧甲基纤维素酶酶活)的测定方法(CMC糖化力法):在容积为1.5mL的离心管中,pH 4.8,50mM的柠檬酸缓冲液(体积为50μL)(一水柠檬酸210g,无菌水750mL,加入NaOH(50-60g),稀释至1000ml,获得pH 4.5的1mol/L柠檬酸缓冲液。当稀释至50mM时,pH值为4.8),置入2%CMC-Na(羧甲基纤维素钠)溶液150μL和适当稀释的酶液50μL,50℃恒温水浴30min。加入1mol/L NaOH溶液50μL和150μL DNS溶液,沸水浴10min,冷水冷却。加入850μL ddH2O,OD540nm下测定光吸收值,测算酶活力。每个处理设置3次重复,灭活酶液作为对照。内切酶酶活力按照国际单位定义为:在pH 4.8,50℃下,每分钟催化水解生成1μmol葡萄糖的酶量为一个酶活力单位IU。
3)β-葡萄糖苷酶酶活力测定:1.5mL的离心管中,加入适当稀释的酶液50μL和柠檬酸缓冲液250μL(浓度为50mM,pH 4.6),50℃恒温水浴10min。加入250μL,50℃预热10min5mM的pNPG溶液(精确称取对硝基苯基-β-D-吡喃半乳糖苷(pNPG)0.3013g,置于100mL容量瓶中,加50mM柠檬酸缓冲液(制备方法同上,pH 4.6)定容至100mL,获得10mM的pNPG溶液,再将其稀释一倍得到5mM的pNPG溶液),50℃恒温水浴10min。加入250μL 1mol/L Na2CO3溶液终止反应,OD410 nm下测定光吸收值,测算酶活力。对照为失活酶液,每个处理设置3次重复。β-葡萄糖苷酶酶活力的定义:在pH 4.6,50℃下,每分钟催化pNPG水解生成1μmol对硝基苯酚(pNP)的酶量为一个单位(IU)。
经测定(如图2所示)盖姆斯木霉TC787的滤纸酶活、内切酶活和β-葡萄糖苷酶活力分别达到2.21、0.41和1.45IU/mL,分别是相同条件下里氏木霉QM9414酶活的2.22倍、0.85倍和3.30倍。表明盖姆斯木霉TC787在实验室条件下液态发酵具有良好的产纤维素酶能力。
实施例3、盖姆斯木霉TC787在固态发酵条件下产滤纸酶酶活和对基物降解能力的测试
分别将水稻秸秆、玉米秸秆、小麦秸秆、浒苔、悬铃木叶片和羊茅草粉碎后过50目筛,分别得到水稻秸秆粉、玉米秸秆粉、小麦秸秆粉、浒苔粉、悬铃木叶片粉和羊茅草粉这6种基质粉。固体基物酶诱导培养基:由固体基物与基础营养盐培养基按照1:2.5的质量比混合高压灭菌得到的培养基,其中,基础营养盐培养基(1L)的组成为:(NH4)2SO4 0.50g/L,KH2PO4 3.0g/L,MgSO4·7H2O 0.50g/L,CaCl2 0.50g/L,其余为水。其中,固体基物由基质粉(分别为水稻秸秆粉、玉米秸秆粉、小麦秸秆粉、浒苔粉、悬铃木叶片粉和羊茅草粉中的任一种)与麸皮比按照1:1的质量比混合而成。分别得到水稻秸秆粉酶诱导培养基(图3和图4中简称水稻秸秆粉)、玉米秸秆粉酶诱导培养基(图3和图4中简称玉米秸秆粉)、小麦秸秆粉酶诱导培养基(图3和图4中简称小麦秸秆粉)、浒苔粉酶诱导培养基(图3和图4中简称浒苔粉)、悬铃木叶片粉酶诱导培养基(图3和图4中简称悬铃木叶片粉)和羊茅草粉酶诱导培养基(图3和图4中简称羊茅草粉)。
1)菌株活化:将斜面保存的盖姆斯木霉TC787在PDA平板上活化,25℃培养3–5d,待其产生大量的分生孢子。孢子悬液的制备:参试菌株在PDA培养基上恒温25℃培养7d,利用无菌蒸馏水冲洗孢子,移液枪将其转移到10mL灭菌离心管中,震荡摇匀。计数,调整孢子浓度至107cfu/mL,作为种子培养液,备用。
2)固态摇瓶发酵:将种子培养液分别转移至上述固体基物酶诱导培养基(水稻秸秆粉酶诱导培养基、玉米秸秆粉酶诱导培养基、小麦秸秆粉酶诱导培养基、浒苔粉酶诱导培养基、悬铃木叶片粉酶诱导培养基和羊茅草粉酶诱导培养基)中,孢子含量均调整为107cfu/g(每克基质中的菌落形成单位,即孢子数目),接种后,8层纱布封口以保证通气量,以100r/min摇动三角瓶,防止培养基板结影响通气,将培养瓶置于摇床30℃培养,分别得到水稻秸秆粉固体曲、玉米秸秆粉固体曲、小麦秸秆粉固体曲、浒苔粉固体曲、悬铃木叶片粉固体曲和羊茅草粉固体曲6种固体曲。这6种固体曲是培养容器内的所有物质,该培养物含有盖姆斯木霉TC787和盖姆斯木霉TC787的代谢物。
3)粗酶液制备:向上述每一种固体曲中加入蒸馏水50mL,40℃摇床浸提1h,过滤,滤液12000rpm离心15min,去除孢子,取上清液(粗酶液)放置10min后待测。
4)滤纸酶酶活性测定:同实施例2。
5)失重率测定:失重率以质量分数ωx计,数值以百分数表示,按照式(1)计算秸秆失重率:ωx=(N0-NX)/N0*100%,式中:NO—腐解前秸秆的干重,克(g);NX—某腐解时间秸秆的干重,克(g)。3次测算取算数平均值作为结果,结果精确至小数点后两位。
盖姆斯木霉TC787在水稻秸秆、玉米秸秆、浒苔、小麦秸秆、羊茅草和悬铃木叶片的诱导下能够产纤维素酶,发酵6天,滤纸酶活分别为2.02、1.89、2.51、5.78、0.39和3.08IU/g干物质(如图3所示)。TC787对6种基物的降解率(失重率)分别为21.9%、24.1%、57.0%、18.3%、13.0%和12.3%(如图4所示)。在固态发酵条件下,盖姆斯木霉TC787表现出良好的基物降解能力和产纤维素酶性能。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
序列表
<110> 中国科学院微生物研究所
<120> 一株高产纤维素酶的盖姆斯木霉及其应用
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 602
<212> DNA
<213> 盖姆斯木霉(Trichoderma gamsii)
<400> 1
ggggggggac agcggaggga tcattaccga gtttacaact cccaaaccca atgtgaacca 60
taccaaactg ttgcctcggc ggggtcacgc cccgggtgcg tcgcagcccc ggaaccaggc 120
gcccgccgga gggaccaacc aaactctttt ctgtagtccc ctcgcggacg ttatttctta 180
cagctctgag caaaaattca aaatgaatca aaactttcaa caacggatct cttggttctg 240
gcatcgatga agaacgcagc gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat 300
catcgaatct ttgaacgcac attgcgcccg ccagtattct ggcgggcatg cctgtccgag 360
cgtcatttca accctcgaac ccctccgggg ggtcggcgtt ggggatcggg aacccctaag 420
acgggatccc ggccccgaaa tacagtggcg gtctcgccgc agcctctact gcgcagtagt 480
ttgcacaact cgcaccggga gcgcggcgcg tccacgtccg taaaacaccc aacttctgaa 540
atgttgacct cggatcaggt aggaataccc gctgaactta agcatatcaa taagcggagg 600
aa 602
<210> 2
<211> 1364
<212> DNA
<213> 盖姆斯木霉(Trichoderma gamsii)
<400> 2
tcgagaaagt tcgagaaggt aagctaattt cactattttt atcactacgc tttattggca 60
cagtcgtgcg tccgacaatc ctgttctcag tcttgtcaat ttttctctcg catcgtcaca 120
ccccgctcta cctgtctcta cccctccttt ggcacagcaa aaattttctg gctgccttgt 180
ttggttttta gtggggtgcc agcttttttt tttctggcaa ccccgctaat cgccgctgtc 240
cctcatccat cgtcttaaca atttgttcac tcaatcgcat ctcattttct ccgtggttca 300
atgtgctgat catgattcaa tcaataggaa gccgccgaac tcggcaaggg ttctttcaag 360
tatgcgtggg ttcttgacaa gctcaaggcc gagcgtgagc gtggtatcac catcgacatt 420
gccctctgga agttcgagac tcccaagtac tatgtcaccg tcattggtat gttttagtat 480
cctcattggc gtttcgaaat catgattcta acgtgccact ctacagacgc tcccggccac 540
cgtgatttca tcaagaacat gatcactggt acctcccagg ctgactgcgc tatcctgatt 600
atcgctgccg gtactggtga gttcgaggct ggtatctcca aggatggcca gacccgtgag 660
cacgctcttc tcgcctacac cctgggtgtc aagcagctca tcgttgccat caacaagatg 720
gacactgcca actgggctga ggctcgttac cttgagatca tcaaggagac ctccaacttc 780
atcaagaagg tcggcttcaa ccccaagacc gttgccttcg tccccatctc cggcttcaac 840
ggtgacaaca tgttggccgc ctccaccaac tgcccctggt acaagggctg ggagaaggag 900
accaaggctg gcaagtccac cggcaagacc cttctcgagg ccattgacgc cattgagccc 960
cccaagcgtc ccacagacaa gccccttcgt ctgccccttc aggatgttta caagatcggt 1020
ggtatcggaa cagtccctgt cggccgtatc gagactggta tcctcaagcc cggtatggtc 1080
gttaccttcg ctccctccaa cgtcaccact gaagtcaagt ccgttgagat gcaccacgag 1140
cagctcgttg agggtgtccc cggtgacaac gttggtttca acgtcaagaa cgtctccgtc 1200
aaggatatcc gccgtggtaa cgttgccggt gactccaaga acgacccccc catgggtgcc 1260
gcttctttca acgcccaggt catcgtcatg aaccaccctg gccaggtcgg tgccggatac 1320
gctcccgtcc tcgattgcca caccgcccac attgcttgca agtt 1364
Claims (10)
1.盖姆斯木霉,其特征在于:所述盖姆斯木霉为盖姆斯木霉(Trichoderma gamsii),其菌株号为TC787,其在中国微生物菌种保藏管理委员会普通微生物中心的登记入册编号为CGMCC No.22416。
2.用于制备纤维素酶的培养物,其特征在于:所述培养物含有权利要求1所述的盖姆斯木霉。
3.如权利要求2所述的培养物,其特征在于:所述培养物是将所述盖姆斯木霉在微生物培养基中培养得到的物质。
4.权利要求1所述的盖姆斯木霉或权利要求2或3所述的培养物在制备纤维素酶中的应用。
5.一种制备纤维素酶的方法,其特征在于:该方法是利用权利要求1所述的盖姆斯木霉进行发酵得到纤维素酶。
6.如权利要求5所述的方法,其特征在于:所述纤维素酶为内切型葡聚糖酶、外切型葡聚糖酶和/或β-葡萄糖苷酶。
7.如权利要求6所述的方法,其特征在于:所述发酵包括用培养基培养所述盖姆斯木霉,所述培养基为液体培养基或固体培养基;所述固体培养基含有基质粉,所述基质粉来源于浒苔、水稻秸秆、玉米秸秆、小麦秸秆、羊茅草和悬铃木叶片中的至少一种。
8.如权利要求7所述的方法,其特征在于:所述固体培养基由固体基物与基础营养盐培养基组成,所述固体培养基中,所述固体基物与基础营养盐培养基的质量比为1:2.5;1L所述基础营养盐培养基的组成为:(NH4)2SO4 4.9-5.1g/L,KH2PO4 2.9-3.1g/L,MgSO4·7H2O0.49-0.51g/L,CaCl2 0.49-0.51g/L,其余为水;所述固体基物由所述基质粉与麸皮组成,所述固体基物中,所述基质粉与所述麸皮的质量比为1:1。
9.权利要求1所述的盖姆斯木霉或权利要求2或3所述的培养物在降解纤维素中的应用。
10.如权利要求9所述的应用,其特征在于:所述纤维素来源于下述至少一种:浒苔、水稻秸秆、玉米秸秆、小麦秸秆、羊茅草和悬铃木叶片。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210718406.8A CN114958623B (zh) | 2022-06-23 | 2022-06-23 | 一株高产纤维素酶的盖姆斯木霉及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210718406.8A CN114958623B (zh) | 2022-06-23 | 2022-06-23 | 一株高产纤维素酶的盖姆斯木霉及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114958623A true CN114958623A (zh) | 2022-08-30 |
CN114958623B CN114958623B (zh) | 2024-03-15 |
Family
ID=82965122
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210718406.8A Active CN114958623B (zh) | 2022-06-23 | 2022-06-23 | 一株高产纤维素酶的盖姆斯木霉及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114958623B (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109852554A (zh) * | 2018-12-25 | 2019-06-07 | 山东大学 | 一株内切葡聚糖酶和β-葡萄糖苷酶双高分泌的里氏木霉工程菌及其构建方法与应用 |
CN113151006A (zh) * | 2021-03-29 | 2021-07-23 | 中国科学院天津工业生物技术研究所 | 能够生产活性提高的纤维素酶的里氏木霉菌株及其应用 |
-
2022
- 2022-06-23 CN CN202210718406.8A patent/CN114958623B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109852554A (zh) * | 2018-12-25 | 2019-06-07 | 山东大学 | 一株内切葡聚糖酶和β-葡萄糖苷酶双高分泌的里氏木霉工程菌及其构建方法与应用 |
CN113151006A (zh) * | 2021-03-29 | 2021-07-23 | 中国科学院天津工业生物技术研究所 | 能够生产活性提高的纤维素酶的里氏木霉菌株及其应用 |
Non-Patent Citations (4)
Title |
---|
RAMIYA BASKARAN ET AL: "Enhanced production of cellulase from a novel strain Trichoderma gamsii M501 through response surface methodology and its application in biomass saccharification", PROCESS BIOCHEMISTRY, vol. 99, pages 48 - 60, XP086379579, DOI: 10.1016/j.procbio.2020.08.006 * |
SAILENDRA GOYARI ET AL: "Population, diversity and characteristics of cellulolytic microorganisms from the Indo-Burma Biodiversity hotspot", SPRINGERPLUS, vol. 700, no. 3, pages 2 - 9 * |
王雪等: "利用玉米秸秆产油脂木霉菌株的筛选", 中国酿造, vol. 31, no. 5, pages 154 - 158 * |
陈凯等: "一株盖姆斯木霉TW20050 的鉴定及功能评价", 科学技术与工程, vol. 16, no. 18, pages 156 - 170 * |
Also Published As
Publication number | Publication date |
---|---|
CN114958623B (zh) | 2024-03-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN102559506B (zh) | 一种草酸青霉菌及其应用 | |
CN102676445B (zh) | 木霉菌剂的制备方法 | |
US20220033762A1 (en) | Penicillium oxalicum SDF-25 strain and application thereof | |
CN103740601B (zh) | 一种高产饲用复合酶的黑曲霉及其应用 | |
CN111073839A (zh) | 暹罗芽孢杆菌、菌剂及其应用 | |
CN101864366B (zh) | 一种桔青霉菌株及其应用 | |
CN116676201A (zh) | 一种波兰青霉cfp-2及其应用 | |
CN105087399A (zh) | 一种秸秆还田纤维分解性生防真菌及菌剂和应用 | |
CN111154661B (zh) | 复合菌剂及其应用 | |
CN114958613B (zh) | 一种螺旋木霉rs05及其在防治麻竹褐腐病中的应用 | |
CN104805029B (zh) | 一种微生态肥的制备方法 | |
CN112940948B (zh) | 一种长枝木霉菌及其应用 | |
CN114958623B (zh) | 一株高产纤维素酶的盖姆斯木霉及其应用 | |
CN1225172C (zh) | 一种大批量生产生物除草剂的方法 | |
CN111575194B (zh) | 一种对秸秆降解的混合菌及其应用 | |
CN112226380B (zh) | 一株降解纤维素生防芽孢杆菌及其应用和制剂 | |
KR20090093545A (ko) | 항 푸른곰팡이 신규 바실러스 푸밀루스, 상기 바실러스푸밀루스가 함유된 분말제제 및 상기 바실러스 푸밀루스가함유되어 배양된 느타리버섯 재배용 발효 폐면배지의제조방법 | |
CN110835610B (zh) | 一种适用于降解秸秆的复合菌剂及制备方法 | |
CN113481111A (zh) | 一种高效生物秸秆发酵菌剂及其制备方法 | |
CN110438026A (zh) | 一种解淀粉芽孢杆菌gld-191及其菌剂、制备方法和应用 | |
CN105176836B (zh) | 一种双出小克银汉霉wu-18菌株及用途 | |
CN104805028A (zh) | 一种微生态肥 | |
CN117535156B (zh) | 一种篮状菌cft-1及其应用 | |
CN110129211B (zh) | 一株耐高温的烟曲霉菌株23#及其应用 | |
CN117384799B (zh) | 一株鲁戈斯芽孢杆菌A78.1、菌剂、产β-葡萄糖苷酶的方法和应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |