CN104328056A - Trichoderma reesei capable of producing cellulase in high yield and application of trichoderma reesei - Google Patents
Trichoderma reesei capable of producing cellulase in high yield and application of trichoderma reesei Download PDFInfo
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- CN104328056A CN104328056A CN201410597983.1A CN201410597983A CN104328056A CN 104328056 A CN104328056 A CN 104328056A CN 201410597983 A CN201410597983 A CN 201410597983A CN 104328056 A CN104328056 A CN 104328056A
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- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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Abstract
The invention provides a trichoderma reesei mutant strain VLKDN-1. The preservation number of the stain VLKDN-1 is CCTCC NO:M 2014506. A trichoderma reesei mutant strain capable of producing cellulase in high yield is obtained by adopting an ultraviolet-induced mutation technology combined with shake flask selection. Compared with a starting strain, the enzyme activity of the fermentation supernatant of mutant strain trichoderma reesei VLKDN-1 is increased by 235%, the content of protein is increased by 157%, the colonial morphology of the mutant strain is obviously smaller than that of the starting strain; the hyphae are shorter than that of the starting strain; the branches are more than that of the starting strain. The viscosity of a fermentation strain solution is remarkably reduced by virtue of the characteristics of short hyphae and more branches of the small colonial morphology of the mutant strain in the production process; the purposes of reducing the stirring rate and improving the dissolved oxygen are achieved; the production cost is reduced; the trichoderma reesei mutant strain VLKDN-1 is wide in application prospect.
Description
Technical field
The invention belongs to microbe to screen technical field, be specifically related to a kind of Trichodermareesei of High Cellulase Production and the application in cellulase production thereof.
Background technology
Trichodermareesei (Trichoderma reesei) is a kind of filamentous fungus, belong to cellulous eukaryotic microorganisms, be the anamorph of Hypocrea jecorina (Hypocrea jecorina), classification be under the jurisdiction of Mycophyta, Deuteromycotina, hyphomycetes, Moniliales, Trichoderma.Trichodermareesei is distributed widely in nature, rotten wood, seed, plant residue, fertilizer, soil and or even air in (Montenecourt & Eveleigh, 1979, Adv Chem Ser.).
Trichodermareesei can express multiple born of the same parents' outer fiber element enzyme and hemicellulase, comprises exocellulase (CBH1 and CBH2), endo cellulase (EG1, EG2, EG3, EG4 and EG5 etc.), beta-glucuroide, zytase and mannase etc.By the synergy of these cellulases and hemicellulase, Mierocrystalline cellulose is thoroughly decomposed into monose (Biely & Tenkanen.1998, In Trichoderma and Gliocladium), therefore, wooden mould cellulase-producing be two generation Mierocrystalline cellulose bio-ethanol Major Enzymes preparation source.
But, trichoderma cellulase and hemicellulase coordinate expression often, and the factor affecting trichoderma cellulase expression of enzymes is a lot.First, cultivate the impact of carbon source: Mierocrystalline cellulose, lactose and sophorose are used for inducing wooden mould cellulase-producing; Glucose and glycerine etc. check the expression of cellulase.The inducing action of carbon source be regulatory factor to affect expression, the positive regulatory factor as relevant to abduction delivering is ACEII and XYR1, and negative regulatory factor is CRE1 and ACEI (Ilme ' n et al., 1997, Mol.Gen.Genet.; Foreman et al., 2003, J.Biol.Chem.).Secondly, thalline (mycelia) form is also one of key factor affecting secreting, expressing.It is generally acknowledged, filamentous fungus is secreted by top and exports extracellular protein.Therefore, multi-branched often on efficient secretion strain morphology.
But it is often very low to produce enzyme level from the trichoderma strain that occurring in nature directly screens, and is not suitable for commercial production.Therefore, how its extracellular protein content is improved, to reduce production cost just one of study hotspot becoming this area by prior art transformation trichoderma strain.
Summary of the invention
The object of this invention is to provide Li's Trichoderma strains and the application thereof of a plant height cellulase-producing, little and the Trichodermareesei mutant strain of High Cellulase Production of a strain colonial morphology is obtained by the method for ultraviolet mutagenesis, the production of cellulase can be widely used in, reduce production cost.
One aspect of the present invention provides a strain Trichodermareesei (Trichoderma reesei) mutant strain VLKDN-1, be preserved in the China typical culture collection center of Wuhan, China Wuhan University on October 22nd, 2014, preserving number is CCTCC NO:M 2014506.
Present invention also offers the application of above-mentioned Trichodermareesei mutant strain VLKDN-1 in cellulase production.
The present invention utilizes ultraviolet mutagenesis technology, and screens the Trichodermareesei mutant strain of acquisition one plant height cellulase-producing in conjunction with shaking flask.Compared with the bacterium that sets out, the fermented supernatant fluid enzyme of mutant bacteria Trichodermareesei VLKDN-1 is lived and is improve 235%, and protein content improves 157%; The colonial morphology of this mutant bacteria is significantly less than the bacterium that sets out, and mycelia is shorter than the mycelia of the bacterium that sets out, branch is many.In process of production, the colonial morphology of this mutant strain " miniaturization " and the feature that mycelia is short, branch is many can make the viscosity of zymocyte liquid significantly reduce, reach and reduce the object that stirring velocity improves again dissolved oxygen, thus be conducive to reducing production cost, application prospect is extensive.
Accompanying drawing explanation
Fig. 1: the colonial morphology of set out bacterium and mutant bacteria compares, wherein: A is the bacterium that sets out, B is mutant bacteria Trichodermareesei VLKDN-1;
Fig. 2: the hypha form of set out bacterium and mutant bacteria compares, wherein: A is the bacterium that sets out, B is mutant bacteria Trichodermareesei VLKDN-1.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in more detail.But the present invention is not limited by the examples.There is provided the object of embodiment to be to make specification sheets comprehensively thorough, and pass on scope of invention to those skilled in the art comprehensively.Except another definition, all technology used in the present invention and scientific terminology all have the same implication usually understood as persons of ordinary skill in the technical field of the present invention.
The screening of embodiment 1 bacterium colony " miniaturization " mutant strain
1, bacterial classification process:
The bacterial classification Trichodermareesei that sets out (this bacterial strain is that the yellow also an ancient unit of weight of inventor herein screened from the withered bark in Laoshan District forest farm, Qingdao City in June, 2013) is inoculated in PDA agar plate (potato 200-300 gram, glucose 20 grams, agar 15-20 gram, 1000 milliliters, tap water, nature pH) on, 28-30 DEG C of constant temperature culture 1 thoughtful stationary phase.After spore maturation, on flat board, add 5-10ml sterilized water scrub spore, draw spore suspension and count, according to spore content its concentration to 10 of adjustment
5-6individual/ml, then carries out follow-up mutagenic treatment.
2, mutagenesis screening:
By above-mentioned spore suspension, join in empty flat board, and carry out magnetic agitation (20-550rpm), then utilize ultraviolet to aim at flat board and carry out mutagenic treatment, mutagenic condition is: 10-20W ultraviolet lamp, and irradiation distance is 10-20cm, and irradiation time is 3-5min; Then the spore suspension after mutagenesis is coated PDA agar plate under half-light or ruddiness condition; Finally by flat board as 30 DEG C of constant temperature culture, until grow bacterium colony, the time is about 7-10 days.
The mutant bacterium that picking colony form is significantly less than the bacterium that sets out is inoculated into PDA agar plate respectively.
The screening of embodiment 2 High Cellulase Production mutant strain
1,96 deep-well plates screenings are utilized:
In the hole of 96 deep-well plates, add 2-3ml inducing culture, inducing culture (GLC substratum) consist of CaCl
21.125g/L, glucose 10g/L, lactose 20g/L, corn steep liquor 15mlg/L, micro-10ml/L, KH
2pO
420g/L, pH5.0; Its medium trace element (ME) consist of FeSO
4.7H
2o 0.75g/L, ZnSO
4.7H
2o 0.06g/L, CuSO
4.5H
2o 0.012g/L, MnSO
4.H
2o0.053g/L, H
3bO
30.003g/L.Then the mutant strain screened in embodiment 1 is inoculated in each hole respectively, is placed on shaking table, 30 DEG C, 200rpm, cultivate 3 days.After cultivation terminates, centrifuging and taking supernatant carries out Enzyme activity assay and SDS-PAGE electrophoresis detection respectively.According to detected result, filter out enzyme and to live and extracellular protein content is significantly higher than mutant bacteria totally 4 strains of the bacterium that sets out, distinguish called after VLKDN-1, VLKDN-2, VLKDN-3, VLKDN-4.
(1) method of enzyme activity determination: CMC method
(2) principle measured: carboxymethyl cellulose can be degraded into oligosaccharides and monose by cellulase.Have the oligosaccharides of reducing end under neutral and monose under boiling water bath condition can with 3,5-dinitrosalicylic acid (DNS) reagent generation color reaction.The reducing sugar amount that the degree of depth and the enzymolysis of reaction solution color produce is directly proportional, and the growing amount of reducing sugar is directly proportional to the vigor of reaction solution cellulase.Therefore, by the intensity of spectral colorimetric assaying reaction liquid color, the vigor of reaction solution cellulase can be calculated.
(3) process is measured: get three test tubes and respectively add 0.5mlCMC substrate, 50 DEG C of water-bath preheating 5min together with enzyme liquid to be measured.0.5ml liquid to be measured is respectively added in first and second test tube, and timing, react 15min in 50 DEG C of water-baths.In three test tubes, respectively add the DNS reagent of 1.5ml after having reacted, and in the 3rd test tube, add the enzyme liquid to be measured of 0.5ml.Take out and after shaking up three test tubes, in boiling water bath, react 5min.Be cooled to room temperature rapidly, be settled to 5.0ml with water.With the absorbancy that the 3rd test tube liquid is to recording first and second test tube liquid under impinging upon 540nm wavelength condition.Enzyme activity value is calculated according to pre-rendered typical curve.
(4) enzyme activity calculates:
Enzyme activity (IU/ml or IU/g)=(value/180/15/0.5 such as glucose) × n
In formula: 180-glucose is converted into micromole from microgram
The reaction times of 15-liquid to be measured and substrate
0.5-add the enzyme liquid measure to be measured of reaction
The extension rate of n-enzyme sample
2, shaking flask screening:
Above-mentioned four plant mutant bacterium (VLKDN-1, VLKDN-2, VLKDN-3, VLKDN-4) are inoculated in (containing 50mlGLC inducing culture) in 500ml triangular flask respectively, are placed on shaking table, 30 DEG C, 200rpm, cultivate 5-7 days.After cultivation terminates, centrifuging and taking supernatant respectively, carry out Enzyme activity assay (CMC Enzyme activity assay method) and determining the protein quantity (Xylene Brilliant Cyanine G detection method), result is as shown in table 1.
| Bacterial strain | Enzyme lives (U/ml) | Protein content (g/L) |
| Set out bacterium | 91 | 0.41 |
| Mutant bacteria VLKDN-1 | 169 | 0.70 |
| Mutant bacteria VLKDN-2 | 143 | 0.60 |
| Mutant bacteria VLKDN-3 | 149 | 0.62 |
| Mutant bacteria VLKDN-4 | 157 | 0.65 |
From the results shown in Table 1, in the 4 plant mutant bacterium that the present invention screens VLKDN-1 fermenting enzyme live and extracellular protein content the highest, than setting out, bacterium improves 70%-85%, achieves significant technique effect.
Applicant is by this mutant strain called after Trichodermareesei VLKDN-1 (Trichoderma reesei VLKDN-1), and the China typical culture collection center of Wuhan, China Wuhan University is preserved on October 22nd, 2014, deposit number is CCTCC NO:M 2014506.
The purifying of embodiment 3 mutant bacteria Trichodermareesei VLKDN-1
Trichodermareesei VLKDN-1 is inoculated on PDA agar plate, 28-30 DEG C of constant temperature culture 1 thoughtful stationary phase.After spore maturation, on flat board, add 5-10ml sterilized water scrub spore; And then draw appropriate spore suspension and coat PDA flat board, finally by flat board as 30 DEG C of constant temperature culture, until grow single bacterium colony; Picking list bacterium colony, namely the mutant bacteria Trichodermareesei VLKDN-1 of purifying, is inoculated in PDA flat board.Meanwhile, to set out bacterium as a control group, adopt above-mentioned same operation to carry out cultivating, purifying, picking list colony inoculation is dull and stereotyped in PDA.
After PDA flat board is cultivated 1 week, the colonial morphology of set out bacterium and mutant bacteria Trichodermareesei VLKDN-1 and the equal significant difference of hypha form.As shown in Figure 1, the colonial morphology of mutant bacteria Trichodermareesei VLKDN-1 is significantly less than the bacterium that sets out; As shown in Figure 2, the mycelia of mutant bacteria Trichodermareesei VLKDN-1 is shorter than the mycelia of the bacterium that sets out, branch is many.
The fermentation scale-up of embodiment 4 mutant bacteria Trichodermareesei VLKDN-1 is cultivated
To set out bacterium and mutant bacteria Trichodermareesei VLKDN-1 is inoculated in shake-flask seed substratum (glucose 10-30g/L respectively, potato 100-200g/L), 30 DEG C, after 48h cultivated by 200rpm shaking table, then (substratum is: glucose 30-50g/L fermented liquid to be proceeded to 7.5L fermentor tank, lactose 2.0-10g/L, corn steep liquor 20-50g/L, ammonium sulfate 10-30g/L, magnesium sulfate 5-10g/L, potassium primary phosphate 15-30g/L), temperature controls at 25 ± 1 DEG C, pH value controls 5.0 ± 0.2, after fermentor cultivation 10h to 15h, start to add lactose-induced thalline and produce enzyme, fermentation time is about 160h to 170h, make zymocyte liquid.
By centrifugal for above-mentioned zymocyte liquid, get supernatant, carry out Enzyme activity assay (CMC Enzyme activity assay method) and determining the protein quantity (Xylene Brilliant Cyanine G detection method).Result shows, and the fermented supernatant fluid enzyme of the bacterium that sets out is lived as 457U/mL, and protein content is 2.47g/L, the fermented supernatant fluid enzyme of mutant bacteria Trichodermareesei VLKDN-1 is lived as 1533U/mL, improve 235% than the bacterium that sets out, protein content is 6.35g/L, and than setting out, bacterium improves 157%.
To sum up, the present invention is by the screening of ultraviolet mutagenesis combine with technique shaking flask, the mutant bacteria Trichodermareesei VLKDN-1 obtained significantly can not only improve the output of cellulase, and the colonial morphology of its " miniaturization " and mycelia is short, branch is many feature can make the viscosity of its zymocyte liquid significantly reduce, reach and reduce the object that stirring velocity improves again dissolved oxygen, thus be conducive to reducing production cost, application prospect is extensive.
Claims (5)
1. a strain Li's Trichoderma strains, is characterized in that, the deposit number of described bacterial strain is CCTCC NO:M 2014506.
2. the preparation method of Li's Trichoderma strains according to claim 1, is characterized in that, described preparation method is obtained by ultraviolet mutagenesis.
3. the application of Li's Trichoderma strains according to claim 1 in cellulase production.
4. prepare a method for cellulase, it is characterized in that, described method is inoculated in fermention medium with Li's Trichoderma strains according to claim 1, adds lactose-induced thalline and carry out fermentative production cellulase after cultivating.
5. method as claimed in claim 4, is characterized in that, described fermention medium composed as follows: glucose 30-50g/L, lactose 2.0-10g/L, corn steep liquor 20-50g/L, ammonium sulfate 10-30g/L, magnesium sulfate 5-10g/L, potassium primary phosphate 15-30g/L.
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Cited By (11)
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| CN104593477A (en) * | 2015-02-13 | 2015-05-06 | 江西省科学院微生物研究所 | Method for quickly separating and screening cellulose decomposing fungi |
| CN105462947A (en) * | 2015-12-31 | 2016-04-06 | 中国科学院天津工业生物技术研究所 | Method for producing cellulose through duckweed |
| CN107603884A (en) * | 2016-07-12 | 2018-01-19 | 青岛蔚蓝生物集团有限公司 | One plant height produces the trichoderma reesei mutant strain of neutral cellulase |
| CN107760606A (en) * | 2016-08-18 | 2018-03-06 | 青岛蔚蓝生物集团有限公司 | A kind of trichoderma reesei mutant strain and its application |
| CN108251310A (en) * | 2016-12-29 | 2018-07-06 | 青岛蔚蓝生物集团有限公司 | A kind of novel reesei host cell and its application |
| CN109988715A (en) * | 2017-12-29 | 2019-07-09 | 青岛蔚蓝生物集团有限公司 | A kind of mutant strain of high yield zytase and its application |
| CN110205250A (en) * | 2019-07-03 | 2019-09-06 | 上海中溶科技有限公司 | One plant of cellulase high-yield and its screening technique and application |
| CN112795491A (en) * | 2021-01-29 | 2021-05-14 | 武汉新华扬生物股份有限公司 | Fermentation method for producing high-activity acidic cellulase by trichoderma reesei |
| CN113717864A (en) * | 2018-01-30 | 2021-11-30 | 青岛蔚蓝生物集团有限公司 | Cellulase high-yield strain and application thereof |
| CN114107359A (en) * | 2022-01-28 | 2022-03-01 | 中国农业科学院北京畜牧兽医研究所 | Method for improving cellulase expression capability of trichoderma reesei by regulating cell metabolism |
| CN114990102A (en) * | 2022-04-29 | 2022-09-02 | 广西大学 | Compound mutation breeding method for cellulase-producing trichoderma strains |
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| CN104593477A (en) * | 2015-02-13 | 2015-05-06 | 江西省科学院微生物研究所 | Method for quickly separating and screening cellulose decomposing fungi |
| CN104593477B (en) * | 2015-02-13 | 2016-08-03 | 江西省科学院微生物研究所 | A kind of sharp separation screen fibre element decomposes the method for fungus |
| CN105462947A (en) * | 2015-12-31 | 2016-04-06 | 中国科学院天津工业生物技术研究所 | Method for producing cellulose through duckweed |
| CN105462947B (en) * | 2015-12-31 | 2019-03-22 | 中国科学院天津工业生物技术研究所 | Utilize the method for duckweed production cellulase |
| CN107603884A (en) * | 2016-07-12 | 2018-01-19 | 青岛蔚蓝生物集团有限公司 | One plant height produces the trichoderma reesei mutant strain of neutral cellulase |
| CN107760606A (en) * | 2016-08-18 | 2018-03-06 | 青岛蔚蓝生物集团有限公司 | A kind of trichoderma reesei mutant strain and its application |
| CN107760606B (en) * | 2016-08-18 | 2020-04-14 | 青岛蔚蓝生物集团有限公司 | Trichoderma reesei mutant strain and application thereof |
| CN108251310A (en) * | 2016-12-29 | 2018-07-06 | 青岛蔚蓝生物集团有限公司 | A kind of novel reesei host cell and its application |
| CN108251310B (en) * | 2016-12-29 | 2020-12-01 | 青岛蔚蓝生物集团有限公司 | Novel trichoderma host cell and application thereof |
| CN109988715A (en) * | 2017-12-29 | 2019-07-09 | 青岛蔚蓝生物集团有限公司 | A kind of mutant strain of high yield zytase and its application |
| CN109988715B (en) * | 2017-12-29 | 2022-03-01 | 青岛蔚蓝生物集团有限公司 | Mutant strain for high-yield xylanase and application thereof |
| CN113717864A (en) * | 2018-01-30 | 2021-11-30 | 青岛蔚蓝生物集团有限公司 | Cellulase high-yield strain and application thereof |
| CN110205250A (en) * | 2019-07-03 | 2019-09-06 | 上海中溶科技有限公司 | One plant of cellulase high-yield and its screening technique and application |
| CN112795491A (en) * | 2021-01-29 | 2021-05-14 | 武汉新华扬生物股份有限公司 | Fermentation method for producing high-activity acidic cellulase by trichoderma reesei |
| CN114107359A (en) * | 2022-01-28 | 2022-03-01 | 中国农业科学院北京畜牧兽医研究所 | Method for improving cellulase expression capability of trichoderma reesei by regulating cell metabolism |
| CN114107359B (en) * | 2022-01-28 | 2022-04-08 | 中国农业科学院北京畜牧兽医研究所 | Method for improving cellulase expression capability of trichoderma reesei by regulating cell metabolism |
| CN114990102A (en) * | 2022-04-29 | 2022-09-02 | 广西大学 | Compound mutation breeding method for cellulase-producing trichoderma strains |
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