CN102925365A - Trichoderma atroviride strain and application thereof in preparation of cellulase - Google Patents
Trichoderma atroviride strain and application thereof in preparation of cellulase Download PDFInfo
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- CN102925365A CN102925365A CN2012104080186A CN201210408018A CN102925365A CN 102925365 A CN102925365 A CN 102925365A CN 2012104080186 A CN2012104080186 A CN 2012104080186A CN 201210408018 A CN201210408018 A CN 201210408018A CN 102925365 A CN102925365 A CN 102925365A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 108010059892 Cellulase Proteins 0.000 title claims description 32
- 229940106157 cellulase Drugs 0.000 title claims description 32
- 241000894120 Trichoderma atroviride Species 0.000 title abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 70
- 102000004190 Enzymes Human genes 0.000 claims abstract description 70
- 239000007788 liquid Substances 0.000 claims abstract description 45
- 238000000855 fermentation Methods 0.000 claims abstract description 31
- 230000004151 fermentation Effects 0.000 claims abstract description 31
- 240000008042 Zea mays Species 0.000 claims abstract description 28
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 28
- 239000007787 solid Substances 0.000 claims abstract description 13
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims abstract description 11
- 235000009973 maize Nutrition 0.000 claims abstract description 11
- 230000007062 hydrolysis Effects 0.000 claims abstract description 10
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 10
- 239000000843 powder Substances 0.000 claims abstract description 10
- 239000002994 raw material Substances 0.000 claims abstract description 7
- 244000005700 microbiome Species 0.000 claims abstract description 5
- 244000068988 Glycine max Species 0.000 claims abstract description 3
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 3
- 229940088598 enzyme Drugs 0.000 claims description 68
- 241000223259 Trichoderma Species 0.000 claims description 34
- 239000010902 straw Substances 0.000 claims description 32
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 17
- 230000001580 bacterial effect Effects 0.000 claims description 17
- 235000005822 corn Nutrition 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 16
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 claims description 15
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 12
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims description 12
- 238000011218 seed culture Methods 0.000 claims description 12
- 239000001888 Peptone Substances 0.000 claims description 11
- 108010080698 Peptones Proteins 0.000 claims description 11
- 241000209140 Triticum Species 0.000 claims description 11
- 235000021307 Triticum Nutrition 0.000 claims description 11
- 238000013016 damping Methods 0.000 claims description 11
- 239000012530 fluid Substances 0.000 claims description 11
- 235000019319 peptone Nutrition 0.000 claims description 11
- 239000002054 inoculum Substances 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 10
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 10
- 235000007164 Oryza sativa Nutrition 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 9
- 235000009566 rice Nutrition 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000000725 suspension Substances 0.000 claims description 8
- 241000609240 Ambelania acida Species 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 6
- 244000046109 Sorghum vulgare var. nervosum Species 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 6
- 239000010905 bagasse Substances 0.000 claims description 6
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 6
- 239000008112 carboxymethyl-cellulose Substances 0.000 claims description 6
- 239000010903 husk Substances 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- 239000011591 potassium Substances 0.000 claims description 6
- 229910052700 potassium Inorganic materials 0.000 claims description 6
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 6
- 235000015099 wheat brans Nutrition 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- 239000001110 calcium chloride Substances 0.000 claims description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 5
- 239000004323 potassium nitrate Substances 0.000 claims description 5
- 235000010333 potassium nitrate Nutrition 0.000 claims description 5
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 5
- 239000011343 solid material Substances 0.000 claims description 4
- 238000010563 solid-state fermentation Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 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 claims description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 238000010298 pulverizing process Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 2
- 239000004202 carbamide Substances 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 2
- 239000011790 ferrous sulphate Substances 0.000 claims description 2
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 2
- 239000007974 sodium acetate buffer Substances 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims 1
- 235000011130 ammonium sulphate Nutrition 0.000 claims 1
- 229920002678 cellulose Polymers 0.000 abstract description 15
- 239000001913 cellulose Substances 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 15
- 238000004519 manufacturing process Methods 0.000 abstract description 10
- 238000004321 preservation Methods 0.000 abstract description 2
- 239000002154 agricultural waste Substances 0.000 abstract 1
- 239000002440 industrial waste Substances 0.000 abstract 1
- 238000009629 microbiological culture Methods 0.000 abstract 1
- 239000002002 slurry Substances 0.000 abstract 1
- 238000000034 method Methods 0.000 description 13
- 108010085318 carboxymethylcellulase Proteins 0.000 description 10
- 102000006995 beta-Glucosidase Human genes 0.000 description 8
- 108010047754 beta-Glucosidase Proteins 0.000 description 8
- 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
- 241000209094 Oryza Species 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 241000499912 Trichoderma reesei Species 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000012978 lignocellulosic material Substances 0.000 description 3
- 231100000219 mutagenic Toxicity 0.000 description 3
- 230000003505 mutagenic effect Effects 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- WTLKTXIHIHFSGU-UHFFFAOYSA-N 2-nitrosoguanidine Chemical compound NC(N)=NN=O WTLKTXIHIHFSGU-UHFFFAOYSA-N 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000218657 Picea Species 0.000 description 1
- 241000351396 Picea asperata Species 0.000 description 1
- 241000124033 Salix Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a trichoderma atroviride B-8-1-34 strain capable of highly yielding cellulose and application of the trichoderma atroviride B-8-1-34 strain in the preparation of the cellulose, belonging to the technical field of microorganisms. The trichoderma atroviride B-8-1-34 strain disclosed by the invention has been preserved in China General Microbiological Culture Collection Center on Aug.10, 2012, with the preservation number of CGMCC No.6390. The trichoderma atroviride B-8-1-34 disclosed by the invention can be used for producing the cellulose by making use of lignocellulose-containing industrial and agricultural wastes in combination of low-cost raw materials such as bran, maize slurry and soybean cake powder through liquid or solid fermentation, has the advantages of short fermentation period, high activity of produced cellulose, reasonable components and high lignocellulose hydrolysis, and provides a novel strain resource to solve the problems on high production cost, low saccharifying efficiency and the like caused by low enzyme activity and unreasonable enzyme components in the utilization of cellulose resources.
Description
Technical field
The present invention relates to dark green trichoderma B-8-1-34 bacterial strain and the application aspect the preparation cellulase thereof of a plant height cellulase-producing, belong to microbial technology field.
Background technology
Cellulose substances is one of the abundantest renewable resources of occurring in nature.Natural cellulose is degraded to available carbohydrate; be further converted to again the materials such as ethanol, tropina, geseous fuel; the problems such as environmental pollution, food shortage, resource anxiety and energy dilemma that the solution world today is faced have Great significance, and cellulase is the most effective biological catalyst of degraded cellulose.
The expensive of enzymolysis process is one of industrialized main restricting factor of biomass energy.Reduce the cellulase cost and relate generally to problems such as reducing cellulase producing bacteria substratum and fermentation costs, raising cellulase activity, therefore, provide and can utilize cheap raw material, the short bacterial strain generation high active cellulase of fermentation period is converted into biomass energy to lignocellulose and has great importance.
Cellulase is of a great variety, and many microorganisms particularly fungi have the ability that produces this prozyme, and wherein enzymatic productivity is stronger that wood is mould, aspergillus, head mold and mould etc., especially in the majority with the Trichoderma bacterial classification.The wooden mould stronger cellulolytic ability that has is grown fast in xylogen, the abundant matrix of Mierocrystalline cellulose.Document " Mutagenesis seed selection cellulase high-yield " (chemistry and the biotechnology such as Hu Tingting, 2008,3, reported take viride as starting strain that 67-67) through mutagenic obtained mutant strain UVT12, its CMC enzyme work reaches 1020.24U/g.Chinese invention patent CN200810023480.8 " a kind of trichoderma reesei cultivation method that improves yield of cellulase ", CN201010040047.2 " a kind of method of High-efficient Production cellulase ", CN200910153354.9 " utilizes Trichodermareesei to prepare cellulase " and CN 201010176883.3 " a kind of liquid fermentation process that improves the cellulase units activity " discloses method and the technique of utilizing Trichodermareesei production of cellulose enzyme; Patent of invention CN200710180525.8 " a kind of production method of acidic liquid cellulase ", CN200910210154.2 " a kind of method of producing liquid cellulase take papermaking short fiber as carbon source through fermentation " and CN200910259677.6 " a kind of preparation method of liquid cellulase " disclose method and the technique of utilizing viride production of cellulose enzyme.Document "
Trichoderma atrovirideMutants with enhanced production of cellulase and β-glucosidase on pretreated willow " (Krisztina Kov á cs etc.; Enzyme and Microbial Technology; 2008; 43:48 – 55) reported that filter paper enzyme activity reaches respectively 1.14,1.18 and 1.14 FPU/mL by the mutagenic obtained dark green trichoderma TUB of three strains F-1721, TUB F-1741 and TUB F-1753.Document " Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with
Trichoderma atrovirideEnzymes produced in-house (Krisztina Kov á cs etc., Biotechnology for Biofuels, 2009,2:14) reported with dark green trichoderma TUB F-1663 production of cellulose enzyme, take wheat stalk as the substrate saccharification, its percent hydrolysis reaches 60-65%, glucose content reaches 9.6～10.4 g/L, document " Enzymatic hydrolysis and simultaneous saccharification and fermentation of steam-pretreated spruce using crude
Trichoderma reeseiAnd
Trichoderma atrovirideEnzymes " (Krisztina Kov á cs etc., Process Biochemistry, 2009,44:1323 – 1329) carry out saccharification take the dragon spruce wood chip as substrate, and its conversion coefficient has reached 62%, and glucose content reaches 18.5 g/L.But also has certain distance apart from industrial applications.
Summary of the invention
The present invention is by screening, and the dark green trichoderma B-8-1-34(that provides a strain enzyme activity high is provided purpose
Trichoderma atrovirideB8-1-34) bacterial strain; Another purpose is to provide the application of this bacterial strain aspect the production of cellulose enzyme.
For realizing the object of the invention, the present invention is take xylose residue as sole carbon source, obtained the stronger bacterial strain B-8 of cellulase-producing ability from the nature separation screening and through ultraviolet ray and nitrosoguanidine mutagenesis seed selection, through Molecular Identification, combining form feature determine this bacterial strain be dark green trichoderma (
Trichoderma atroviride), further obtain a plant height through mutagenic and breeding and produce the dark green trichoderma B-8-1-34 of bacterial strain.In preservation on August 10 in 2012, preserving number was CGMCC No. 6390 to this bacterial strain.
The application of this bacterial strain aspect the preparation cellulase, realize by liquid state fermentation:
⑴ after number be the dark green trichoderma B-8-1-34 activation of CGMCC No. 6390 with culture presevation, making concentration was 10
6～10
8The spore suspension of/mL, in 1～10% inoculum size access seed culture medium, 26～32 ℃, 150～220 rpm shaking culture get seed liquor, then seed liquor are accessed in the liquid fermentation medium with 2～15% inoculum sizes, initial pH4.0～6.0, liquid amount is 30～60mL/250mL, in 26～32 ℃, cultivates 60～96h in 150～220rpm shaking table;
⑵ get supernatant liquor as crude enzyme liquid with the fermented liquid centrifugation that step ⑴ obtains;
Described seed culture medium forms: carboxymethyl cellulose 1%, peptone 0.2%, (NH
4)
2SO
40.3%, MgSO
40.04%, CaCl
20.04%, KH
2PO
40.1%, surplus is distilled water, and each component by weight percentage;
Described liquid fermentation medium forms: single composition or the mixture of 1～5% maize straw, corn cob, straw, rice husk, Wheat Straw, kaoliang stalk, xylose residue, bagasse, and add wheat bran 0.05～3%, corn steep liquor 0.05～5%, the single composition of peptone 0.05～1.0 % or yeast powder 0.05～1.0% or more than one compositions; Add 0.01～0.5% saltpetre, 0.02～0.3% potassium primary phosphate, 0.01～0.08% sal epsom and 0～0.05% calcium chloride; Add manganous sulfate, ferrous sulfate or the cobalt chloride of 0.01～0.2mmol/L, surplus is distilled water, and each component by weight percentage.
The present invention also can realize by solid state fermentation:
(1) with culture presevation number be the dark green trichoderma B-8-1-34 activation of CGMCC No. 6390 after, making concentration is 10
6～10
8The spore suspension of/ml.The spore suspension direct inoculation to solid medium or be seeded to seed culture medium and cultivate, then is seeded to the solid fermentation culture medium culturing with seed liquor with 2～15% inoculum sizes.The bottling amount is 6～12 g/250mL, and solid-liquid ratio is 1:1～2, in 26-32 ℃ of cultivation, and regular stirring.
(2) fermentation 60～120h, add damping fluid lixiviate 0.5～4h, the lixiviate damping fluid is acetic acid-sodium-acetate buffer or the 0.05 mol/L pH5.0 citric acid-sodium citrate damping fluid of 0.05 mol/L pH5.0, its add-on is 8～12 times of solid materials weight, solid-liquid separation prepares crude enzyme liquid, measures its FPA, the work of CMC enzyme and beta-glucosidase enzyme and lives.
Described solid medium is the PDA solid medium;
Described seed culture medium forms: carboxymethyl cellulose 1%, peptone 0.2%, (NH
4)
2SO
40.3%, MgSO
40.04%, CaCl
20.04%, KH
2PO
40.1%, surplus is distilled water.Each component by weight percentage.121 ℃ of autoclaving 25 min in 26～32 ℃, cultivate 18～26h in 150～220rpm shaking table, namely get seed liquor.The seed liquor of above-mentioned preparation is accessed in the fermention medium with 2～15% inoculum sizes.
Described solid fermentation substratum forms: single composition or the mixtures such as maize straw, corn cob, straw, rice husk, Wheat Straw, kaoliang stalk, xylose residue, bagasse, cross 40～60 mesh sieves after the raw material pulverizing, addition is 10～60%, be preferably 20% xylose residue and 40% straw, add wheat bran (2～40%), corn steep liquor (5～40%), soybean cake powder (0.5～5%), (NH4)
2SO
4One or more mixtures of (0.3～3%), saltpetre (0.01～1.0%), ammonium nitrate (0.01～1.0%), peptone (0.01～1.0%) or urea (0.01～1.0%); Add potassium primary phosphate (0.2～2%), calcium chloride (0.02～0.2%), magnesium sulfate heptahydrate (0.01～0.1%).Each component by weight percentage.
The crude enzyme liquid of liquid fermenting or solid fermentation preparation as mentioned above can be through ultrafiltration, saltout or the method such as organic solvent deposit obtains the cellulase powder.Filter paper enzyme activity (FPA), the work of CMC enzyme and beta-glucosidase enzyme by fermentation and mensuration institute cellulase-producing are lived.
The application of this bacterial strain aspect hydrolyzing ligno-cellulose with cellulosic enzyme:
In the acetic acid-sodium-acetate or citric acid-sodium citrate damping fluid of 0.05mol/L pH 5.0, crude enzyme liquid or the enzyme powder of as above preparation are joined in the lignocelluloses such as maize straw, corn cob, straw, rice husk, Wheat Straw, kaoliang stalk, xylose residue, bagasse 45～55 ℃ of hydrolysis 24～120h.Solid-liquid ratio is 0.5～2:10, and the enzyme addition is 1～6FPA/ml.Lignocellulose and enzyme can also can add in batches in disposable adding.
The dark green trichoderma B-8-1-34(of a strain provided by the invention
Trichoderma atrovirideB-8-1-34), it is liquid to produce in enzymic fermentation substratum when take 2.5% xylose residue as carbon source, fermentation 66h, and FPA reaches 176.32IU/g, and the CMC enzyme is lived and is that it is 206.72IU/g that 238.4IU/g, beta-glucosidase enzyme live.With crude enzyme liquid saccharification xylose residue and corn cob 48h, concentration of reduced sugar reaches respectively 3.7% and 4.4%.It is advantageous that fermentation period is short, institute's cellulase-producing vigor is high, and component is reasonable, and the hydrolysis of lignocellulose ability is strong.Enzyme activity is not high in the cellulose resource utilization, the unreasonable production cost that causes of enzyme component is high in order to solve in the present invention, the problems such as saccharification efficient is low provide new microorganism resource, be conducive to suitability for industrialized production, lignocellulose be converted into biomass energy have great importance.
This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 10th, 2012, and ((No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No. 6390.
Embodiment
Enzyme is produced in embodiment 1 dark green trichoderma B-8-1-34 liquid state fermentation
To make 10 after the culture presevation number dark green trichoderma B-8-1-34 bacterial strain activation for CGMCC No. 6390
6The mL spore suspension, in 5% inoculum size access seed culture medium, in 30 ℃, cultivate 24h in the 180rpm shaking table, again so that (liquid amount 40 mL/250mL), initial pH is 4.0～6.0 in the 10% inoculum size access liquid culture medium, in 30 ℃, be cultured in the 200rpm shaking table at 66 o'clock, the centrifugal 10min of 4000r/min, preparation crude enzyme liquid.
Described seed culture medium forms: carboxymethyl cellulose 1%, peptone 0.2%, (NH
4)
2SO
40.3%, MgSO
40.04%, CaCl
20.04%, KH
2PO
40.1%, surplus is distilled water.Each component by weight percentage.
Described liquid fermentation medium forms: 2.5% maize straw, corn cob, Wheat Straw or xylose residue and straw 0.5%, and add wheat bran (1.5%), corn steep liquor (4%), peptone (0.1 %); Add 0.3% saltpetre, 0.15% potassium primary phosphate, 0.04% sal epsom; Add the manganous sulfate of 0.01～0.2mmol/L, surplus is distilled water.Each component by weight percentage.
Mensuration FPA, CMC enzyme are lived, the beta-glucosidase enzyme is lived.Enzyme activity determination uses the DNS method.With at 50 ℃, under pH 5.0 conditions, the hydrolysis of 1min catalytic substrate produces the reducing sugar that is equivalent to 1 μ mol glucose and is defined as an international enzyme activity unit, is converted into the IU/g solid material and represents.
Dark green trichoderma B-8-1-34 utilizes different lignocellulosic material liquid state fermentation cellulase-producing results as shown in table 1.
The enzyme result is produced in the dark green trichoderma B-8-1-34 liquid state fermentation of table 1
| Raw material (2.5%) | FPA（IU/g） | CMCase（IU/g） | Beta-glucosidase (IU/g) |
| Maize straw | 56.20 | 56.04 | 36.44 |
| Corn cob | 48.92 | 77.04 | 59.08 |
| Wheat Straw | 35.22 | 56.96 | 38.88 |
| Xylose residue | 176.32 | 238.4 | 206.72 |
Embodiment 2 dark green trichoderma B-8-1-34 Produced by Solid-state Fermentation enzymes
Be 10 with the concentration of making after the culture presevation number dark green trichoderma strain activation for CGMCC No. 6390
8In the spore suspension access seed culture medium of/ml, in 30 ℃, cultivate 24h in the 180rpm shaking table, be connected to the solid fermentation culture medium culturing with 3% inoculum size again, regular stirring, adds the citric acid-sodium citrate damping fluid of 0.05 mol/L pH5.0 of 10 times of volumes by 30 ℃ when being cultured to 94h, 150rpm shakes lixiviate, and 4000r/min is centrifugal, and 10min prepares crude enzyme liquid.
Described seed culture medium forms: carboxymethyl cellulose 1%, peptone 0.2%, (NH
4)
2SO
40.3%, MgSO
40.04%, CaCl
20.04%, KH
2PO
40.1%, surplus is distilled water.Each component by weight percentage.
Described solid fermentation substratum forms: the single composition of maize straw, corn cob, straw, Wheat Straw or xylose residue or mixture 60%, cross 40～60 mesh sieves after the raw material pulverizing, add wheat bran 37.7%, add potassium primary phosphate 2%, calcium chloride (0.2%), magnesium sulfate heptahydrate (0.1%).Each component by weight percentage.
Measuring crude enzyme liquid FPA, the work of CMC enzyme and beta-glucosidase enzyme lives.Enzyme activity determination adopts the DNS method.With at 50 ℃, under the condition of pH 5.0, the hydrolysis of 1min catalytic substrate produces the reducing sugar that is equivalent to 1 μ mol glucose and is defined as an international enzyme activity unit, is converted into the IU/g solid material and represents.
Dark green trichoderma B-8-1-34 utilizes different lignocellulosic material solid fermentation cellulase-producing results as shown in table 2.
The dark green trichoderma B-8-1-34 Produced by Solid-state Fermentation enzyme result of table 2
| Raw material (60%) | FPA（IU/g） | CMCase（IU/g） | Beta-glucosidase enzyme (IU/g) alive |
| Maize straw | 6.94 | 10.87 | 8.81 |
| Corn cob | 4.22 | 9.18 | 7.64 |
| Wheat Straw | 4.89 | 11.62 | 11.53 |
| Rice straw | 7.04 | 11.64 | 10.42 |
| 40% rice straw+20% xylose residue | 6.86 | 12.58 | 16.46 |
The application of embodiment 3 saccharification lignocelluloses
In the acetic acid-sodium-acetate or citric acid-sodium citrate damping fluid of 0.05mol/L pH 5.0, crude enzyme liquid or the enzyme powder of as above preparation are joined in corn cob, rice straw powder or the xylose residue, solid-liquid ratio is 1:10,50 ℃, measure concentration of reduced sugar and glucose concn behind the 180rpm hydrolysis 48h, the results are shown in Table 3.。The enzyme addition is 4FPA/ml.Lignocellulose and enzyme can also can add in batches in disposable adding.
Table 3 saccharification lignocellulose result
Annotate: reducing sugar test adopts the DNS method; Glucose assays adopts enzyme process (SBA-40c type bio-sensing analyser)
Embodiment 4 temperature of reaction, pH are on the impact of cellulase activity and heat and the ph stability of enzyme
Under 40 ° of C, 45 ° of C, 50 ° of C, 55 ° of C, measure respectively FPA and the CMCase of crude enzyme liquid, live the highest measured value as with reference to (100%) take enzyme, calculate relative enzyme and live.After placing respectively 45 ° of C, 50 ° of C, 55 ° of C, 60 ° of C, 65 ° of C to be incubated 3 h the enzyme liquid, measure FPA and CMCase, calculate residual enzyme live (seeing Table 4).The result shows that the enzyme reaction optimum temperuture is about 45 ℃, and 50 ° of C are better with stability inferior.
Table 4 temperature is on enzyme activity impact and Thermostability
| Temperature (° C) | 40 | 45 | 50 | 55 | 60 | 65 |
| Relative enzyme (FPA) alive | 78% | 100% | 75% | 70% | / | / |
| Relative enzyme (CMCase) alive | 82% | 100% | 78% | 65% | / | / |
| Residual enzyme (FPA) alive | / | 98% | 98% | 37% | 30% | 8% |
| Residual enzyme (CMCase) alive | / | 98% | 98% | 55% | 42% | 12% |
Use respectively pH 4.5,5.0,5.5,6.0 citrate buffer solution, under the suitableeest temperature of reaction, measure FPA and the CMCase of crude enzyme liquid, live the highest measured value as with reference to (100%) take enzyme, calculate relative enzyme and live.Enzyme liquid is mixed with pH 4.5,5.0,5.5,6.0 and 6.5 damping fluid respectively, behind 50 ° of C insulation 3h, measure FPA and CMCase, calculate residual enzyme live (seeing Table 5).The result shows that the optimal pH of enzyme reaction is about 5.0, and is best in pH about 5.0 stability.
Table 5 pH is on the pH stability of enzyme activity impact and enzyme
| pH | 4.5 | 5.0 | 5.5 | 6.0 | 6.5 |
| Relative enzyme (FPA) alive | 79% | 100% | 63% | 59% | / |
| Relative enzyme (CMCase) alive | 58% | 100% | 42% | 40% | / |
| Residual enzyme (FPA) alive | 62% | 96% | 39% | 25% | 8% |
| Residual enzyme (CMCase) alive | 75% | 96% | 59% | 50% | 14% |
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (5)
1. the dark green trichoderma B-8-1-34(of a strain
Trichoderma atrovirideB-8-1-34) bacterial strain is characterized in that, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No. 6390.
2. utilize preserving number as claimed in claim 1 for the dark green trichoderma B-8-1-34 bacterial strain of CGMCC No. 6390 in the application aspect the preparation cellulase, it is characterized in that, realize by liquid state fermentation:
⑴ after number be the dark green trichoderma B-8-1-34 activation of CGMCC No. 6390 with culture presevation, making concentration was 10
6～10
8The spore suspension of/mL, in 1～10% inoculum size access seed culture medium, 26～32 ℃, 150～220 rpm shaking culture get seed liquor, then seed liquor are accessed in the liquid fermentation medium with 2～15% inoculum sizes, initial pH4.0～6.0, liquid amount is 30～60mL/250mL, in 26～32 ℃, cultivates 60～96h in 150～220rpm shaking table;
⑵ get supernatant liquor as crude enzyme liquid with the fermented liquid centrifugation that step ⑴ obtains;
Described seed culture medium forms: carboxymethyl cellulose 1%, peptone 0.2%, (NH
4)
2SO
40.3%, MgSO
40.04%, CaCl
20.04%, KH
2PO
40.1%, surplus is distilled water, and each component by weight percentage;
Described liquid fermentation medium forms: single composition or the mixture of 1～5% maize straw, corn cob, straw, rice husk, Wheat Straw, kaoliang stalk, xylose residue, bagasse, and add wheat bran 0.05～3%, corn steep liquor 0.05～5%, the single composition of peptone 0.05～1.0 % or yeast powder 0.05～1.0% or more than one compositions; Add 0.01～0.5% saltpetre, 0.02～0.3% potassium primary phosphate, 0.01～0.08% sal epsom and 0～0.05% calcium chloride; Add manganous sulfate, ferrous sulfate or the cobalt chloride of 0.01～0.2mmol/L, surplus is distilled water, and each component by weight percentage.
3. utilize preserving number as claimed in claim 1 for the dark green trichoderma B-8-1-34 bacterial strain of CGMCC No. 6390 in the application aspect the preparation cellulase, it is characterized in that, realize by solid state fermentation:
(1) with culture presevation number be the dark green trichoderma B-8-1-34 activation of CGMCC No. 6390 after, making concentration is 10
6～10
8The spore suspension of/ml to solid medium or be seeded to seed culture medium, in 26～32 ℃, is cultivated 18～26h in 150～220rpm shaking table with the spore suspension direct inoculation, gets seed liquor; Then seed liquor is seeded to the solid fermentation culture medium culturing with 2～15% inoculum sizes; The bottling amount is 6～12 g/250mL, and solid-liquid ratio is 1:1～2, in 26-32 ℃ of cultivation, and regular stirring;
(2) fermentation 60～120h, add damping fluid lixiviate 0.5～4h, the lixiviate damping fluid is acetic acid-sodium-acetate buffer or the 0.05 mol/L pH5.0 citric acid-sodium citrate damping fluid of 0.05 mol/L pH5.0, its add-on is 8～12 times of solid materials weight, and solid-liquid separation prepares crude enzyme liquid;
Described seed culture medium forms: carboxymethyl cellulose 1%, peptone 0.2%, (NH
4)
2SO
40.3%, MgSO
40.04%, CaCl
20.04%, KH
2PO
40.1%, surplus is distilled water, and each component by weight percentage;
Described solid fermentation substratum forms: single composition or the mixture of maize straw, corn cob, straw, rice husk, Wheat Straw, kaoliang stalk, xylose residue, bagasse, cross 40～60 mesh sieves after the raw material pulverizing, and addition is 40～60%; Add one or more mixtures of wheat bran 2～40%, corn steep liquor 5～40%, soybean cake powder 0.5～5%, ammonium sulfate 0.3～3%, saltpetre 0.01～1.0%, ammonium nitrate 0.01～1.0%, peptone 0.01～1.0% or urea 0.01～1.0%; Add potassium primary phosphate 0.2～2%, calcium chloride 0.02～0.2%, magnesium sulfate heptahydrate 0.01～0.1%, each component is by weight percentage.
4. utilize preserving number as claimed in claim 1 for the dark green trichoderma B-8-1-34 institute cellulase-producing of CGMCC No. 6390 in the application aspect the hydrolysis of lignocellulose, it is characterized in that, in the acetic acid-sodium-acetate or citric acid-sodium citrate damping fluid of 0.05mol/L pH 5.0, be that crude enzyme liquid or the enzyme powder of the dark green trichoderma B-8-1-34 institute cellulase-producing of CGMCC No. 6390 joins in the lignocellulose with preserving number, 45～55 ℃ of hydrolysis 24～120h, solid-liquid ratio is 0.5～2:10, and the enzyme addition is 1～6FPA/mL.
5. the application of the dark green trichoderma B-8-1-34 of utilization as claimed in claim 4 institute's cellulase-producing aspect hydrolysis of lignocellulose, it is characterized in that described lignocellulose is one or more of maize straw, corn cob, straw, rice husk, Wheat Straw, kaoliang stalk, xylose residue, bagasse.
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| CN104845953A (en) * | 2015-05-22 | 2015-08-19 | 徐州工程学院 | Method for preparing complex enzyme preparation of heat-resistant ferulic acid esterase and cellulose by fermenting trichoderma atroviride through airlift fermentation tank |
| CN104893988A (en) * | 2015-05-22 | 2015-09-09 | 徐州工程学院 | Trichoderma atroviride strain capable of producing high-temperature-resistant feruloyl esterase and high-temperature-resistant cellulase and application thereof |
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| CN104893988A (en) * | 2015-05-22 | 2015-09-09 | 徐州工程学院 | Trichoderma atroviride strain capable of producing high-temperature-resistant feruloyl esterase and high-temperature-resistant cellulase and application thereof |
| CN104893988B (en) * | 2015-05-22 | 2017-08-25 | 徐州工程学院 | A kind of Trichoderma atroviride and its application for producing high temperature resistant feruloyl esterase and high temperature-resisting cellulase |
| CN104845953B (en) * | 2015-05-22 | 2017-12-19 | 徐州工程学院 | A kind of method that Trichoderma atroviride airlift fermentor fermentation prepares heat-resisting feruloyl esterase and cellulase complex enzyme formulation |
| CN105567577A (en) * | 2016-01-29 | 2016-05-11 | 广西中烟工业有限责任公司 | Trichoderma atroviride strain and application thereof |
| CN105567577B (en) * | 2016-01-29 | 2019-08-23 | 广西中烟工业有限责任公司 | A kind of Trichoderma atroviride strain and its application |
| CN112662651A (en) * | 2021-01-12 | 2021-04-16 | 武汉科技大学 | Method for producing cellulase and grease by using nitrogen-rich biomass |
| CN112662651B (en) * | 2021-01-12 | 2023-03-03 | 武汉科技大学 | Method for producing cellulase and grease by using nitrogen-rich biomass |
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| CN113475352B (en) * | 2021-07-21 | 2023-02-24 | 嘉兴学院 | Seedling raising substrate and preparation method thereof |
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