CN103740600A - Strain for producing cellulases - Google Patents
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Abstract
The invention belongs to the field of microbial fermentation, and discloses a Trichoderma reesei strain, particularly application of a Trichoderma reesei strain with the collection number of CCTCC No:M2013540 in producing cellulases by fermentation. The optimum pH value of the strain for producing cellulases is 3.0-6.0, and the optimum temperature is 23-35 DEG C. The method comprises the following steps: inoculating a strain seed solution in a fermentation medium, and culturing at 25 DEG C for 104 hours until the activities of the excision beta-glucanase, incision beta-glucanase, beta-glucosaccharase and filter paper enzyme in the fermentation liquid respectively reach 680U/mL, 1389U/mL, 486U/mL and 792U/mL.
Description
Technical field
The invention belongs to microorganism field, particularly a kind of fungi of cellulase-producing.
Background technology:
Cellulase is extensively present in natural organism.Can produce cellulase in bacterium, fungi, animal body etc.The cellulase that is generally used for production comes from fungi, more typically has wooden enzyme to belong to (Trichodema), Aspergillus (Aspergillus) and Penicillium (Penicillium).Cellulase is all widely used in food service industry and environmental industry.When carrying out zymamsis, the interpolation of cellulase can increase the utilization ratio of raw material, and vinosity is promoted to some extent.
Cellulase is of a great variety, originates very wide.Its structure and function of the cellulase of different sources differs greatly.Because fungin production of enzyme is high, activity large, therefore the cellulase of applying in livestock industry and fodder industry is mainly fungal cellulase.
Cellulase can be divided into endoglucanase (1 according to the difference of its catalyzed reaction function, 4-β-D-glucan glucanohydrolase or endo-1,4-β-D-glucanase, EC3.2.1.4), from the abbreviation EG of fungi, from abbreviation Cen, the exoglucanase (1 of bacterium, 4-β-D-glucan cellobilhydrolase or exo-1,4-β-D-glucannase, EC.3.2.1.91), from the abbreviation CBH of fungi, from the abbreviation Cex of bacterium) and beta-glucan glycosides enzyme (β-1,4-glucosidase, EC.3.2.1.21) abbreviation BG.The unformed area of the random cutting fibre element polysaccharide chain of endoglucanase inside, produces the oligosaccharides of different lengths and the end of new chain.Exoglucanase acts on the end of the fibrination sugar chain of these reductibilities and irreducibility, discharges glucose or cellobiose.Beta-glucosidase hydrolysis fiber disaccharides produces bimolecular glucose.Fungin production of enzyme is high, active large, the cellulase in main using fungus source in livestock industry and feed work.
Cellulase reaction and general enzyme reaction are different, and its topmost difference is that cellulase is polycomponent enzyme system, and substrate structure is extremely complicated.Due to the water-insoluble of substrate, the ES mixture process that the adsorption of cellulase has replaced enzyme-to-substrate to form.Cellulase is first adsorbed on substrate Mierocrystalline cellulose specifically, then under the synergy of several components, cellulose decomposition is become to glucose.
Nineteen fifty, Reese etc. have proposed C1-Cx hypothesis, and this hypothesis is thought and must, with different enzyme synergies, Mierocrystalline cellulose could be hydrolyzed to glucose thoroughly.Synergy is commonly considered as the first cellulosic noncrystalline domain of attack of endoglucanase (C1 enzyme), form the required new free-end of Cx, then by CX enzyme, from reducing end or the non-reducing end of polysaccharide chain, cut cellobiose unit, finally by beta-glucan glycosides enzyme, cellobiose is hydrolyzed into two glucose.But, the synergy order of cellulase is not absolute, in research subsequently, finds, C1-Cx and beta-glucan glycosides enzyme must exist simultaneously and could be hydrolyzed natural cellulose.If first use C1 enzyme effect crystalline cellulose, then remove C1 enzyme, then add Cx enzyme, so sequential action but can not be hydrolyzed crystalline cellulose.
Strain improvement is the basic work of cellulase production, domestic and international many experts have carried out large quantity research, in order to produce high-quality cellulase product, Wang Jialin etc. (1996) are absorbing on the basis of domestic and foreign experience, viride wood 10, viride Sn-91014, koning trichoderma NT-15, aspergillus niger XX-15A have successively been introduced, on this basis, ultraviolet ray, specific electromagnetic wave radiation, linear accelerator have been adopted, the mutafacient system of the physics such as nitrosoguanidine, chemistry, has obtained superior strain NT15-H, NT15-H1, XT-15H, XT-15H1.Wherein wooden mould NT-15H solid culture vigor detects and shows through station, China National Light Industrial Products Department food quality supervision inspection center Nanjing, and filter paper vigor is 3670u/g, C1-enzyme activity 24460u/g, and Cx-enzyme activity 1800u/g, reaches advanced world standards.It is stable that this bacterial classification is produced performance in batch production.Zhang Linghua etc. (1998) adopt koning trichoderma W-925, J-931, after over-richness is 2% ethyl sulfate and ultraviolet ray (15W, 30cm, 2min) complex mutation, obtained the high Wu-932 bacterial classification of inulinase-producing activity, this bacterial classification CMC saccharogenic power reaches 2975, filter paper carbohydrase activity is 531, and than setting out, bacterium W-925 has improved respectively 100% and 81%.The king Cheng Shu of feed additive technology service centre of the Ministry of Chemical Industry etc. (1997) adopt the Trichodermareesei A3 at this center first to carry out after ultraviolet ray and nitrosoguanidine complex mutation, by the spore inoculating of processing on fiber double-layer plate, cultivate 5-8 days for 30 ℃, place 7-10 days for 15 ℃, select single bacterium colony that transparent circle diameter and colony diameter are larger and carry out triangular flask solid state fermentation and screen again, obtained the very high Trichodermareesei 91-3 bacterial strain of cellulase-producing vigor.
The production technique of cellulase mainly contains two kinds, i.e. solid fermentation and liquid fermenting, and its technique is as follows:
1, affect the factor of yield of enzyme and vigor: the factor that affects yield of cellulase and vigor is a lot, except bacterial classification, also have culture temperature, pH, moisture, matrix, incubation time etc.These factors do not isolate, but connect each other.Zhang Zhongliang etc. (1997) adopt homogeneous design Cl12 (1210), take viride (T.ViriclePers.expr) as bacterial classification, studied five effects of large factor to yield of enzyme and vigor that affect cellulase-producing, thought that matrix crude fiber content is 40%, initial pH7.5, add 4 times, water, under 26-31 ℃ of condition, cultivate 45h and can obtain maximum yield of enzyme 26mg/g and CMC enzyme activity 20mg/gh.Wang Chenghua etc. (1997) have also studied the condition of enzyme production of the Trichodermareesei 91-3 of its mutagenesis screening, result shows straw powder and the wheat bran of this bacterial classification with 7:3, another interpolation 4% ammonium sulfate, 0.4% potassium primary phosphate, 0.1% magnesium sulfate are optimal medium, 28-32 ℃ is suitable culture temperature, 30 ℃ is optimum temps, 4% is optimum inoculation amount, and 96h arrives fermentation peak.Zhang Linghua etc. (1998) have studied with koning trichoderma W-925 as the bacterium that sets out, the optimal conditions of fermentation of the Wu-932 High-Cellulase-Yielding bacterium obtaining after mutagenesis.Result shows, take the wheat bran of 1:2 and rice straw powder as substratum, and 5% inoculum size, straw is pulverized mean length 3-5mm, initial pH4-5, temperature is at 28-35 ℃, and fermentation time 72h is optimal conditions of fermentation.
Number of patent application is 201010040047, and applicant Zhejiang University the invention provides a kind of method of production of cellulose enzyme, comprises the following steps: by Trichodermareesei (TriCh.Derma reesei) be seeded in fermention medium, after fermentation, start feeding culture, in feeding culture process, when fermented liquid pH value is higher than 4.8 time, fed-batch medium, when fermented liquid pH value is lower than 4.5 time, stop fed-batch medium, total fermentation time reaches 192-240 hour, stops fermentation.The inventive method organically combines insoluble carbon source and solubility carbon source, the expression of co-induction cellulose enzyme gene.By feeding culture, make the growth of bacterial classification and the forming process of object meta-bolites reach coordinated balance, solved merely the problem existing take Mierocrystalline cellulose or soluble sugar in the technique of inductor production of cellulose enzyme, effectively improved the fermentation level of cellulase, and the cellulase obtaining is stronger to the degradation property of cellulosic substrate.
The preparation method of 2,013,102,687,288 1 kinds of low temperature neutral cellulases of application number, described low temperature neutral cellulase is prepared take Trichodermareesei as bacterial strain, this preparation method comprises: activated spawn, ultraviolet mutagenesis, then cultivate at low temperatures, purifying, and purifying bacterial strain is prepared to seed liquor in 10-15 ℃ of bottom fermentation, seed liquor is inoculated in culture medium, at 10-15 ℃, cultivate 96-144 hour, just obtain low temperature neutral cellulase; The present invention also provides a kind of preparation method of described low temperature neutral cellulase.Beneficial effect of the present invention is: simple to operate, fermentation period is short, can reduce the application of temperature of enzyme, thereby reduces the power consumption of industrial application, makes its more environmental protection, has broad application prospects; In addition, the present invention also provides a kind of preparation method of this cellulase, and it can be widely used in the modification of paper-making fibre, reduces the cost of paper industry fibre modification.
Summary of the invention
Technical problem to be solved by this invention is to provide the Li's Trichoderma strains of the high and good stability of a strain cellulase-producing vigor.Trichodermareesei provided by the present invention (Trichodema reesei) 601-17, this bacterial strain is preserved in Chinese Typical Representative culture collection center on November 3rd, 2013, deposit number is CCTCC NO:M2013540, Classification And Nomenclature is Trichodermareesei 601-17Trichodema reesei601-17, preservation address: wuchang, wuhan district Luo Jia Shan Wuhan University Life Science College, postcode 430072.
Described bacterial strain physiological and biochemical property:
This bacterial strain is grown on PDA solid medium, and the colony characteristics of formation is that bacterium colony is flocculence, and bacterium colony is light green, and bacterium colony is flat, high 0.1-0.75mm, and colony edge white, neat; Fast growth, 48h colony diameter reaches 1.0-8.5mm, and 72h reaches 30-50mm; White mycelium, has barrier film, and mycelia wall is smooth, and diameter is at 2-5 micron.Conidiophore occurs from the short lateral branch of mycelia, on side shoot to life.Conidiophore is ampuliform, and upright, colourless, spore is spherical in shape, green, diameter 20-100 micron.
This bacterial strain can be grown on wheat bran, and main metabolites is cellulase (endo cellulase, circumscribed cellulase and glucuroide).According to < < An Introduction industrial mycology > > (George Smith1954), the common and conventional fungus G reatT.GreaT.GT > of < < fungi identification handbook > > (Wei Jing surpasses 1982), < < (institute of microbiology of the Chinese Academy of Sciences 1973), identify that this bacterial strain is: Trichodermareesei.
Cultural characteristic: the optimal pH 3.0-6.0 of this bacterial strain cellulase-producing; Optimum temperuture is 23～35 ℃.
Seed liquor is inoculated in fermention medium, 25 ℃ cultivate 104 hours, fermented liquid cellulase circumscribed beta-glucanase, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity reach respectively 680U/mL, 1389U/mL, 486U/mL and 792U/mL.
Utilize Li's Trichoderma strains, as follows by the method for ultraviolet mutagenesis, cultivation, fermentative production cellulase.
Li's Trichoderma strains is inoculated on slant activation substratum, activated spawn; Cultivate the bacterial classification after activation, picking list bacterium colony is prepared spore suspension, and uses uviolizing spore suspension, and mutagenesis obtains spore bacterium colony;
Spore bacterium colony after mutagenic and breeding is coated in primary dcreening operation substratum, at 20-35 ℃, cultivates 3-5 days, selects this bacterial strain of primary dcreening operation bacterial strain purifying; Purifying bacterial strain is inoculated in fermention medium by 5% inoculum size, and enlarged culturing is prepared seed liquor step by step, and incubation time is 72-96 hour; Seed liquor is inoculated in fermention medium by the inoculum size of fermentating liquid volume 5-10%, cultivates 96-144 hour for 20-35 ℃, and Trichodermareesei fermentative production cellulase finishes; Fermented liquid is centrifugal at 4000-6000rpm, and collecting gained liquid is crude enzyme liquid; The crude enzyme liquid obtaining carries out hyperconcentration filtration, obtains concentrated enzyme liquid.
Beneficial effect of the present invention is: the Li's Trichoderma strains of the high and good stability of the strain cellulase-producing vigor of having obtained, change strain fermentation and produce the acidic cellulase of high vigor, its preparation method is simple, fermentation period is short, can greatly reduce the power consumption of industrial application, make its more environmental protection, there is wide prospects for commercial application.
Embodiment:
Bacterial classification primary dcreening operation: the soil sample screening and separating that original strain Trichodermareesei (Trichodema reesei) HYX01 picks up from Jinshi City guarantor river levee domestic fungus cultivating base goes out.Li's Trichoderma strains is inoculated on slant activation substratum, activated spawn; Cultivate the bacterial classification after activation, picking list bacterium colony is prepared spore suspension, and uses uv irradiating spore suspension, and mutagenesis obtains spore bacterium colony; Suitably dilution is adjusted to 103/mL by spore concentration, gets last dilution bacterium liquid 0.2mL, and dilution spread is on Mierocrystalline cellulose-Congo red plate screening substratum.200 of 30 ℃ of cultivation bacterial strains that after 2～3 days, picking transparent circle/colony diameter is larger.(described Mierocrystalline cellulose-Congo red plate screening substratum is composed as follows: cellulose powder 10g, Congo red 0.2g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, gelatin 2g, agar 20g, tap water constant volume 1000mL, pH value 5-6,121 ℃ of sterilizing 20min).
Multiple sieve: the 200 strain bacterium that obtain are inoculated in to slant medium with aseptic toothpick respectively, and 30 ℃ are cultured to spore and are paved with inclined-plane.Respectively spore is fermented to be inoculated under aseptic washing to be equipped with in the 250mL triangular flask that 50mL sieves substratum again, inoculum size 10%(v/v), 30 ℃, 100r/min are cultivated 96h, measure respectively the cellulase activity of each bacterial strain.(described sieve again substratum composed as follows: cellulose powder 50g, ammonium sulfate 5g, magnesium sulfate 0.25g, potassium primary phosphate 1g, sodium-chlor 0.1g, tap water constant volume 1000mL, pH value 5-6,121 ℃ of sterilizing 20min).Choose the bacterial strain that cellulose enzyme vigor is the highest and carry out amplification test.
Filtering out again bacterial strain is Trichodermareesei (Trichodema reesei) 601-17, and deposit number is CCTCC NO:M2013540.
Cultural characteristic: the optimal pH 3.0-6.0 of this bacterial strain cellulase-producing; Optimum temperuture is 23～35 ℃.
Genetic stability test: this bacterial strain is gone down to posterity for continuous ten times on inclined-plane, and the method for sieving again by shaking flask detects the fermentation situation after at every turn going down to posterity.Experiment discovery is gone down to posterity for continuous ten times on inclined-plane, and this bacterial classification proterties does not have considerable change, and property indices is all normal, illustrates that the genetic stability of this bacterial classification is stronger.
Scale-up
Seed culture: by bacterial strain access 500mL triangular flask the highest cellulose enzyme vigor, 100 milliliters of seed culture medium loading amounts, 30 ℃, 150rpm shaking table cultivation 72-96h., seed culture medium: cellulose powder 5%, ammonium sulfate 0.3%, magnesium sulfate 0.025%, potassium primary phosphate 0.1%, sodium-chlor 0.01%, all the other are water, pH value 5-6,121 ℃ of sterilizing 20min.
Seed tank culture: by seed liquor with 10%(v/v) inoculum size access is equipped with in the 10L fermentor tank of 7.5L fermention medium, controlling pH value constant is 5.0 ± 0.2,27 ± 0.1 ℃ of culture temperature, stirring velocity 300rpm, ventilation (v/v) 1:0.8-1.2, incubation time 104h, dissolved oxygen 20-30%.Described fermention medium preparation method is: cellulose powder 10%, ammonium sulfate 0.5%, magnesium sulfate 0.025%, potassium primary phosphate 0.1%, sodium-chlor 0.01%, all the other are water, pH value 5-6,121 ℃ of sterilizing 20min.
After fermentation ends, get fermented supernatant fluid (crude enzyme liquid) and carry out enzyme activity detection, after measured, circumscribed beta-glucanase, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity reach respectively 680U/mL, 1389U/mL, 486U/mL and 792U/mL.
Bacterial strain after Trichodermareesei (Trichodema reesei) mutagenesis of using in the present invention can be preserved 2 months in 4 ℃ of environment, in the Sorbitol Solution USP of 10-20%, at subzero 80 ℃, can preserve for a long time.
Slant medium: potato 20%, glucose 1%, agar 2%, all the other are water, pH nature, 28 ℃ of temperature.
Claims (4)
1. a bacterial strain for cellulase-producing, is characterized in that, described bacterial strain is Trichodermareesei bacterial classification (Trichoderma reesei) 601-17, and deposit number is CCTCC NO:M2013540.
2. the bacterial strain of cellulase-producing according to claim 1, is characterized in that, described bacterial strain physiological and biochemical property is as follows: this bacterial strain is grown on PDA solid medium, the bacterium colony forming is flocculence, light green, and bacterium colony is flat, high 0.1-0.75mm, colony edge white, neat; Fast growth, 48h colony diameter reaches 1.0-8.5mm, and 72h reaches 30-50mm; White mycelium, has barrier film, and mycelia wall is smooth, and diameter is at 2-5 micron; Conidiophore occurs from the short lateral branch of mycelia, on side shoot to life; Mitogenetic armful of son stalk is ampuliform, and upright, colourless, spore is spherical in shape, green, diameter 20-100 micron.
3. the bacterial strain of cellulase-producing according to claim 1 and 2, is characterized in that, the optimal pH 3.0-6.0 of described bacterial strain cellulase-producing, and optimum temperuture is 23-35 ℃.
4. the bacterial strain of cellulase-producing according to claim 1 and 2, seed liquor is inoculated in fermention medium, 25 ℃ cultivate 104 hours, fermented liquid cellulase circumscribed beta-glucanase, Endo-β-glucanase, beta-glucosidase and filter paper enzyme activity reach respectively 680U/mL, 1389U/mL, 486U/mL and 792U/mL.
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| US14/578,408 US9605247B2 (en) | 2013-12-23 | 2014-12-20 | Strain and a method to produce cellulase and its use |
| US15/360,076 US10053680B2 (en) | 2013-12-23 | 2016-11-23 | Strain and a method to produce cellulase and its use |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104263658A (en) * | 2014-09-26 | 2015-01-07 | 中国科学院天津工业生物技术研究所 | Trichoderma reesei strain and application thereof |
| CN105176833A (en) * | 2015-08-07 | 2015-12-23 | 山东大学 | Trichoderma strain capable of generating cellulase and application of trichoderma strain |
| CN105802854A (en) * | 2014-12-30 | 2016-07-27 | 中国科学院上海生命科学研究院 | Cellulase high-yielding bacterial strain and application thereof |
| CN107012102A (en) * | 2017-01-13 | 2017-08-04 | 东南大学 | The trichoderma reesei genetic engineering bacterium of one plant of High Cellulase Production in the case where soluble and non-solubility carbon source is induced and construction method and application |
| CN110317746A (en) * | 2019-05-31 | 2019-10-11 | 中国农业科学院兰州畜牧与兽药研究所 | One plant of cellulose-degrading bacteria CX10 and its application from termite gut |
| CN111876335A (en) * | 2020-07-31 | 2020-11-03 | 深圳市顺盛农业科技发展有限公司 | Fungus capable of degrading cellulose and separation method and application thereof |
| CN112920973A (en) * | 2021-03-22 | 2021-06-08 | 广西壮族自治区兽医研究所 | Bacillus subtilis GL-4 for producing cellulase and application thereof |
| CN114480137A (en) * | 2021-12-24 | 2022-05-13 | 鞍钢集团矿业有限公司 | Trichoderma reesei and method for degrading straw by same |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263658A (en) * | 2014-09-26 | 2015-01-07 | 中国科学院天津工业生物技术研究所 | Trichoderma reesei strain and application thereof |
| CN105802854A (en) * | 2014-12-30 | 2016-07-27 | 中国科学院上海生命科学研究院 | Cellulase high-yielding bacterial strain and application thereof |
| CN105802854B (en) * | 2014-12-30 | 2020-10-16 | 中国科学院分子植物科学卓越创新中心 | Cellulase high-yield strain and application thereof |
| CN105176833A (en) * | 2015-08-07 | 2015-12-23 | 山东大学 | Trichoderma strain capable of generating cellulase and application of trichoderma strain |
| CN107012102A (en) * | 2017-01-13 | 2017-08-04 | 东南大学 | The trichoderma reesei genetic engineering bacterium of one plant of High Cellulase Production in the case where soluble and non-solubility carbon source is induced and construction method and application |
| CN107012102B (en) * | 2017-01-13 | 2020-06-30 | 东南大学 | Trichoderma reesei genetic engineering bacterium capable of producing cellulase at high yield under induction of soluble and non-soluble carbon sources, and construction method and application thereof |
| CN110317746A (en) * | 2019-05-31 | 2019-10-11 | 中国农业科学院兰州畜牧与兽药研究所 | One plant of cellulose-degrading bacteria CX10 and its application from termite gut |
| CN111876335A (en) * | 2020-07-31 | 2020-11-03 | 深圳市顺盛农业科技发展有限公司 | Fungus capable of degrading cellulose and separation method and application thereof |
| CN112920973A (en) * | 2021-03-22 | 2021-06-08 | 广西壮族自治区兽医研究所 | Bacillus subtilis GL-4 for producing cellulase and application thereof |
| CN112920973B (en) * | 2021-03-22 | 2022-10-28 | 广西壮族自治区兽医研究所 | Bacillus subtilis GL-4 for producing cellulase and application thereof |
| CN114480137A (en) * | 2021-12-24 | 2022-05-13 | 鞍钢集团矿业有限公司 | Trichoderma reesei and method for degrading straw by same |
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