CN102925365B - 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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- CN102925365B CN102925365B CN201210408018.6A CN201210408018A CN102925365B CN 102925365 B CN102925365 B CN 102925365B CN 201210408018 A CN201210408018 A CN 201210408018A CN 102925365 B CN102925365 B CN 102925365B
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- 241000894120 Trichoderma atroviride Species 0.000 title claims abstract description 7
- 108010059892 Cellulase Proteins 0.000 title claims description 32
- 229940106157 cellulase Drugs 0.000 title claims description 32
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 108090000790 Enzymes Proteins 0.000 claims abstract description 71
- 102000004190 Enzymes Human genes 0.000 claims abstract description 71
- 239000007788 liquid Substances 0.000 claims abstract description 45
- 238000000855 fermentation Methods 0.000 claims abstract description 30
- 230000004151 fermentation Effects 0.000 claims abstract description 30
- 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 69
- 241000223259 Trichoderma Species 0.000 claims description 33
- 239000010902 straw Substances 0.000 claims description 32
- 239000000203 mixture Substances 0.000 claims description 25
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 17
- 235000005822 corn Nutrition 0.000 claims description 17
- 230000001580 bacterial effect Effects 0.000 claims description 16
- 239000001963 growth medium 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
- 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
- 229910052799 carbon Inorganic materials 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
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 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
- 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
- 230000004913 activation Effects 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 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
- 229910052943 magnesium sulfate Inorganic materials 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
- 235000019341 magnesium sulphate Nutrition 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
- 239000013028 medium composition Substances 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
- 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
- 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
- 239000000243 solution Substances 0.000 description 3
- 239000002023 wood Substances 0.000 description 3
- 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
- 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
- 241000196324 Embryophyta 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
- 230000003213 activating 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
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 the dark green trichoderma B-8-1-34 bacterial strain of a plant height cellulase-producing and in the application of preparing aspect cellulase, belong to microbial technology field.
Background technology
Cellulose substances is one of renewable resources of enriching the most 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 the 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 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 of this prozyme of generation, and what wherein enzymatic productivity was stronger have, and wood is mould, aspergillus, head mold and mould etc., especially in the majority with Trichoderma bacterial classification.Wood is mould has a stronger cellulolytic ability, and in the abundant matrix of xylogen, Mierocrystalline cellulose, growth is fast.Document " Mutagenesis seed selection cellulase high-yield " (chemistry and the biotechnology such as Hu Tingting, 2008,3,67-67) reported take viride as starting strain, and 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 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) report with dark green trichoderma TUB F-1663 production of cellulose enzyme, take wheat stalk as 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 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 object
trichoderma atrovirideb8-1-34) bacterial strain; Another object is to provide the application of this bacterial strain aspect 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 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 the dark green trichoderma B-8-1-34 of a strain superior strain through mutagenic and breeding.This bacterial strain is in preservation on August 10 in 2012, and preserving number is CGMCC No. 6390.
This bacterial strain, in the application of preparing aspect cellulase, is realized by liquid state fermentation:
(1), after by culture presevation number being the dark green trichoderma B-8-1-34 activation of CGMCC No. 6390, making concentration is 10
6～10
8the spore suspension of/mL, in 1～10% inoculum size access seed culture medium, 26～32 ℃, 150～220 rpm shaking culture, obtain seed liquor, then seed liquor are accessed in liquid fermentation mediums with 2～15% inoculum sizes, initial pH4.0～6.0, liquid amount is 30～60mL/250mL, in 26～32 ℃, in 150～220rpm shaking table, cultivates 60～96h;
(2) the fermented liquid centrifugation (1) step being obtained, gets supernatant liquor as crude enzyme liquid;
Described seed culture medium composition: 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 composition: 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% magnesium sulfate and 0～0.05% calcium chloride; Manganous sulfate, ferrous sulfate or the cobalt chloride of adding 0.01～0.2mmol/L, surplus is distilled water, each component is by weight percentage.
The present invention also can realize by solid state fermentation:
(1) after by culture presevation number being the dark green trichoderma B-8-1-34 activation of CGMCC No. 6390, making concentration is 10
6～10
8the spore suspension of/ml.Spore suspension is directly inoculated on solid medium or is seeded to seed culture medium and cultivate, then seed liquor is seeded to solid fermentation culture medium culturing with 2～15% inoculum sizes.Bottling amount is 6～12 g/250mL, and solid-liquid ratio is 1:1～2, in 26-32 ℃ of cultivation, and regularly stirring.
(2) fermentation 60～120h, add damping fluid lixiviate 0.5～4h, 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 is prepared crude enzyme liquid, measures its FPA, the work of CMC enzyme and beta-glucosidase enzyme and lives.
Described solid medium is PDA solid medium;
Described seed culture medium composition: 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, obtain seed liquor.The seed liquor of above-mentioned preparation is accessed in fermention mediums with 2～15% inoculum sizes.
Described solid fermentation substratum composition: single composition or the mixtures such as maize straw, corn cob, straw, rice husk, Wheat Straw, kaoliang stalk, xylose residue, bagasse, after raw material pulverizing, cross 40～60 mesh sieves, 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 that as mentioned above prepared by liquid fermenting or solid fermentation, can be through ultrafiltration, saltout or the method such as organic solvent deposit obtains cellulase powder.Live and beta-glucosidase enzyme is lived by the filter paper enzyme activity (FPA), CMC enzyme that ferment and measure institute's cellulase-producing.
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 to 45～55 ℃ of hydrolysis 24～120h.Solid-liquid ratio is 0.5～2:10, and enzyme addition is 1～6FPA/ml.Lignocellulose and enzyme can disposablely add also and can add in batches.
The dark green trichoderma B-8-1-34(of a strain provided by the invention
trichoderma atrovirideb-8-1-34), it is liquid produces in enzymic fermentation substratum when take 2.5% xylose residue as carbon source, fermentation 66h, and FPA reaches 176.32IU/g, and CMC enzyme is lived as 238.4IU/g, and it is 206.72IU/g that beta-glucosidase enzyme is lived.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 hydrolysis of lignocellulose ability is strong.The present invention is that in the utilization of solution cellulose resource, enzyme activity is not high, the unreasonable production cost causing of enzyme component is high, the problems such as saccharification efficiency is low provide new microorganism resource, be conducive to suitability for industrialized production, lignocellulose be converted into biomass energy and have great importance.
This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's 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 the dark green trichoderma B-8-1-34 liquid state fermentation of embodiment 1
After activating, the dark green trichoderma B-8-1-34 bacterial strain that is CGMCC No. 6390 by culture presevation number makes 10
6mL spore suspension, in 5% inoculum size access seed culture medium, in 30 ℃, in 180rpm shaking table, cultivate 24h, then with in 10% inoculum size access liquid culture medium (liquid amount 40 mL/250mL), initial pH is 4.0～6.0, in 30 ℃, in 200rpm shaking table, be cultured at 66 o'clock, the centrifugal 10min of 4000r/min, prepares crude enzyme liquid.
Described seed culture medium composition: 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 composition: 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% magnesium sulfate; The manganous sulfate that adds 0.01～0.2mmol/L, surplus is distilled water.Each component by weight percentage.
Mensuration FPA, CMC enzyme are lived, beta-glucosidase enzyme is lived.Enzyme activity determination uses 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 the international enzyme activity unit of Yi Ge, is converted into 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.
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 |
the dark green trichoderma B-8-1-34 Produced by Solid-state Fermentation enzyme of embodiment 2
The concentration of making after the dark green trichoderma strain that is CGMCC No. 6390 by culture presevation number activates is 10
8in the spore suspension access seed culture medium of/ml, in 30 ℃, in 180rpm shaking table, cultivate 24h, be connected to solid fermentation culture medium culturing with 3% inoculum size again, regularly stirring,, adds the citric acid-sodium citrate damping fluid of 0.05 mol/L pH5.0 of 10 times of volumes by 30 ℃ while being cultured to 94h, 150rpm shakes lixiviate, and 4000r/min is centrifugal, and 10min prepares crude enzyme liquid.
Described seed culture medium composition: 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 composition: the single composition of maize straw, corn cob, straw, Wheat Straw or xylose residue or mixture 60%, after raw material pulverizing, cross 40～60 mesh sieves, add wheat bran 37.7%, add potassium primary phosphate 2%, calcium chloride (0.2%), magnesium sulfate heptahydrate (0.1%).Each component by weight percentage.
Mensuration crude enzyme liquid FPA, CMC enzyme are lived and beta-glucosidase enzyme is lived.Enzyme activity determination adopts 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 the international enzyme activity unit of Yi Ge, is converted into 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 xylose residue, solid-liquid ratio is 1:10,50 ℃, after 180rpm hydrolysis 48h, measure concentration of reduced sugar and glucose concn, the results are shown in Table 3.。Enzyme addition is 4FPA/ml.Lignocellulose and enzyme can disposablely add also and can add in batches.
Table 3 saccharification lignocellulose result
Note: reducing sugar test adopts DNS method; Glucose assays adopts enzyme process (SBA-40c type bio-sensing analyser)
embodiment 4 temperature of reaction, the pH impact on 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.By enzyme liquid be placed in respectively 45 ° of C, 50 ° of C, 55 ° of C, 60 ° of C, 65 ° of C are incubated after 3 h, measure FPA and CMCase, calculate residual enzyme live (in table 4).Result shows that enzyme reaction optimum temperuture is 45 ℃ of left and right, and 50 ° of C are better with stability inferior.
The thermostability of table 4 temperature on enzyme activity impact and enzyme
| Temperature (° C) | 40 | 45 | 50 | 55 | 60 | 65 |
| Enzyme (FPA) alive relatively | 78% | 100% | 75% | 70% | / | / |
| Enzyme (CMCase) alive relatively | 82% | 100% | 78% | 65% | / | / |
| Residual enzyme (FPA) alive | / | 98% | 98% | 37% | 30% | 8% |
| Residual enzyme (CMCase) alive | / | 98% | 98% | 55% | 42% | 12% |
Use respectively the citrate buffer solution of pH 4.5,5.0,5.5,6.0, 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 the damping fluid of pH 4.5,5.0,5.5,6.0 and 6.5 respectively, after 50 ° of C insulation 3h, measure FPA and CMCase, calculate residual enzyme (in table 5) alive.Result shows that the optimal pH of enzyme reaction is 5.0 left and right, best in pH 5.0 left and right stability.
The pH stability of table 5 pH on enzyme activity impact and enzyme
| pH | 4.5 | 5.0 | 5.5 | 6.0 | 6.5 |
| Enzyme (FPA) alive relatively | 79% | 100% | 63% | 59% | / |
| Enzyme (CMCase) alive relatively | 58% | 100% | 42% | 40% | / |
| Residual enzyme (FPA) alive | 62% | 96% | 39% | 25% | 8% |
| Residual enzyme (CMCase) alive | 75% | 96% | 59% | 50% | 14% |
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of 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)
- The dark green trichoderma of one strain ( trichoderma atroviride) B-8-1-34 bacterial strain, it is characterized in that, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC No. 6390.
- Preserving number as claimed in claim 1 be the dark green trichoderma B-8-1-34 bacterial strain of CGMCC No. 6390 in the application of preparing aspect cellulase, it is characterized in that, realize by liquid state fermentation:(1), after by culture presevation number being the dark green trichoderma B-8-1-34 activation of CGMCC No. 6390, making concentration is 10 6～10 8the spore suspension of/mL, in 1～10% inoculum size access seed culture medium, 26～32 ℃, 150～220 rpm shaking culture, obtain seed liquor, then seed liquor are accessed in liquid fermentation mediums with 2～15% inoculum sizes, initial pH4.0～6.0, liquid amount is 30～60mL/250mL, in 26～32 ℃, in 150～220rpm shaking table, cultivates 60～96h;(2) the fermented liquid centrifugation (1) step being obtained, gets supernatant liquor as crude enzyme liquid;Described seed culture medium composition: 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 composition: single composition or the mixture of 1～5% maize straw, corn cob, straw, rice husk, Wheat Straw, kaoliang stalk, xylose residue, bagasse, and add single composition or more than one compositions of wheat bran 0.05～3%, corn steep liquor 0.05～5%, peptone 0.05～1.0 % or yeast powder 0.05～1.0%; Add 0.01～0.5% saltpetre, 0.02～0.3% potassium primary phosphate, 0.01～0.08% magnesium sulfate and 0～0.05% calcium chloride; Manganous sulfate, ferrous sulfate or the cobalt chloride of adding 0.01～0.2mmol/L, surplus is distilled water, each component is by weight percentage.
- Preserving number as claimed in claim 1 be the dark green trichoderma B-8-1-34 bacterial strain of CGMCC No. 6390 in the application of preparing aspect cellulase, it is characterized in that, realize by solid state fermentation:(1) after by culture presevation number being the dark green trichoderma B-8-1-34 activation of CGMCC No. 6390, making concentration is 10 6～10 8the spore suspension of/ml, is directly inoculated on solid medium by spore suspension or is seeded to seed culture medium, in 26～32 ℃, in 150～220rpm shaking table, cultivates 18～26h, obtains seed liquor; Then seed liquor is seeded to solid fermentation culture medium culturing with 2～15% inoculum sizes; Bottling amount is 6～12 g/250mL, and solid-liquid ratio is 1:1～2, in 26-32 ℃ of cultivation, and regularly stirring;(2) fermentation 60～120h, add damping fluid lixiviate 0.5～4h, 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 is prepared crude enzyme liquid;Described seed culture medium composition: 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 composition: single composition or the mixture of maize straw, corn cob, straw, rice husk, Wheat Straw, kaoliang stalk, xylose residue, bagasse, after raw material pulverizing, cross 40～60 mesh sieves, 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. preserving number as claimed in claim 1 to be CGMCC No. 6390 dark green trichoderma B-8-1-34 institute cellulase-producing is in the application aspect 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, crude enzyme liquid or the enzyme powder of to be CGMCC No. 6390 by preserving number dark green trichoderma B-8-1-34 institute cellulase-producing join in lignocellulose, 45～55 ℃ of hydrolysis 24～120h, solid-liquid ratio is 0.5～2:10, and 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 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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| 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 |
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| 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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