CN109988715A - A kind of mutant strain of high yield zytase and its application - Google Patents

A kind of mutant strain of high yield zytase and its application Download PDF

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CN109988715A
CN109988715A CN201711477714.1A CN201711477714A CN109988715A CN 109988715 A CN109988715 A CN 109988715A CN 201711477714 A CN201711477714 A CN 201711477714A CN 109988715 A CN109988715 A CN 109988715A
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zytase
trichoderma reesei
mutant strain
enzyme
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CN109988715B (en
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刘士成
李�瑞
王华明
曹兴南
王贵斌
宋雅丽
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Weifang Kdn Biotech Co ltd
Qingdao Vland Biotech Group Co Ltd
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/885—Trichoderma

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Abstract

The invention belongs to beneficial microbes, and screening technique field is transformed, and in particular to a kind of trichoderma reesei mutant strain of high yield zytase and its application.The deposit number of the mutant strain is CCTCC NO:M2017797.After the mutant strain shake flask fermentation 120h, xylanase activity reaches 1500U/mL in fermented supernatant fluid, and bacterium germination improves 80% more out, and unexpected technical results have been achieved.Trichoderma reesei mutant strain provided by the invention can be widely applied to the fermenting and producing of zytase, advantageously reduce the production cost of the enzyme, promote the popularization and application of zytase.

Description

A kind of mutant strain of high yield zytase and its application
Technical field
The invention belongs to beneficial microbes, and screening technique field is transformed, and in particular to a kind of Richter scale wood of high yield zytase Mould mutant strain and its application.
Background technique
Xylan is the main component of hemicellulose, is the main polysaccharide structure of plant cell, is accounted for all renewable organic The 1/3 of carbon.Xylan is typically found in Plant Secondary Materials wall, the hardwood (cell wall of 15-30% being largely present in angiosperm Ingredient), the cork (cell wall constituent of 7-10%) in gymnosperm and annual plant (< 30% cell wall constituent). In cell wall, xylan is present in the intersection of cellulose and lignin, this for plant cell wall fibrous polymer power and Integrality is particularly significant.
Zytase be it is a kind of can compound enzyme system by xylan degrading at xylo-oligosaccharide or xylose, mainly include inscribe β-l, 4-D- zytase, β-D- xylosidase, α-L- arabinofuranosidase, α-D- glucuronidase, alpha-D-glucose Aldehyde neuraminidase, acetyl xylan esterase and phenolic acid esterase.The degree of polymerization of the zytase of separate sources in main chain, the number of branch Amount, type, length and its binding site are all different.During degradation of xylan, excision enzyme is mutually promoted with restriction endonuclease, Accelerate the degradation process of xylan.
The production of zytase at present mainly has 3 approach: 1) screening can secrete the microorganism of novel hydrolase；2) lead to It crosses enzyme engineering means and current industrial strain is transformed；3) optimize some impact factors in process of production, such as substrate type, culture Condition, enzyme preparation recycle and the redesign of production procedure.
The microorganism that can secrete zytase that can be applied to production is mainly fungi and bacterium, and wherein fungi is mainly wrapped Aspergillus, mould etc. are included, bacterium mainly includes streptomycete and bacillus etc..For example, being point in place of the characteristic of mould Pol6 The optimum reaction conditions for the Extracellular xylanase secreted are acidity, can be applied compared with extreme environment, such as feed and field of food.Meter Qu Zytase caused by mould HML366 has preferable thermal stability and pH tolerance, can be applied to papermaking and bioenergy Field.Zytase secreted by streptomycete CS624 and bacillus pumilus SS1 can efficiently digest wheat bran and generate oligomeric wood Sugar.Different sulphur streptomycete LMZM is carried out liquid fermentation by substrate of corncob, and generated zytase can when bleaching for paper pulp To improve the brightness of paper.The zytase that fibrosis cellulose bacteria CKMX1 is generated is living in pH5.0-9.0,50-60 DEG C of temperature Property stablize, can be widely used in pulping and paper-making field.
With the continuous development of technique for gene engineering, xylanase gene can Escherichia coli, bacillus, yeast, It is expressed in filamentous fungi expression system.For example, split spore bacterium from thermophilic and cloned XynA gene and expressed in pichia pastoris X-33, Recombination zymoprotein can be maintained in pH6.0-9.0 and 60-80 DEG C of environment 60% enzymatic activity, due to its to pH and temperature have compared with High tolerance makes the enzyme have biggish application potential.The xylanase gene Xyn11A being cloned into from bacillus, should Gene encodes 366 amino acid, and the optimal reactive temperature of recombinant protease is 55 DEG C.By the XynB gene of aspergillus niger IA-001 into Row optimization, and be cloned into Pichia pastoris GS115, the recombinant protein after optimization improves 2.8 times than wild type enzyme activities, recombination Albumen afterwards can express higher xylanase activity.
The present invention obtains the production bacterial strain that xylanase activity improves by the method screening of mutagenesis, industrialized to adapt to Large-scale production, and promote the further development of zytase.
Summary of the invention
Gather the object of the present invention is to provide a kind of trichoderma reesei (Trichoderma reesei) mutant strain and its in wood Application in carbohydrase production.The mutant strain that applicant is obtained by the method screening of ultraviolet mutagenesis, can greatly improve xylan The expression quantity of enzyme can be widely applied to the production of zytase, reduce the cost of the enzyme, be conducive to answering extensively for zytase With.
One aspect of the present invention provides a kind of mutant strain trichoderma reesei Mu17 (Trichoderma reesei Mu17), It is preserved in the China typical culture collection center of Wuhan, China Wuhan University on December 15th, 2017, deposit number is CCTCC NO:M2017797。
One aspect of the present invention provides application of the trichoderma reesei in production zytase.
It is using the trichoderma reesei as fermentation strain the present invention also provides a kind of method for producing zytase.
It is to be fermented to obtain by the trichoderma reesei the present invention also provides a kind of zytase.
The present invention is screened by ultraviolet mutagenesis method using trichoderma reesei Mu as starting strain and obtains mutant strain trichoderma reesei Mu17.After the mutant strain shake flask fermentation 120h, xylanase activity reaches 1500U/mL in fermented supernatant fluid, relatively sets out Bacterium improves 80%, and unexpected technical results have been achieved.The mutant bacteria Mu17 is compared with bacterium germination out to be had in bacterium colony phenotype Apparent variation, the bacterium colony of mutant bacteria Mu17 obviously become smaller, and colony diameter is only the 1/2 of bacterium germination, and bacterium colony is more closely knit, favorably In reducing zymocyte liquid viscosity, the dissolved oxygen content in fermentation process is improved, and then is conducive to improve biomass, increases foreign protein Secreting, expressing amount.Trichoderma reesei mutant strain provided by the invention can be widely applied to the fermenting and producing of zytase, favorably In the production cost for reducing the enzyme, promote the popularization and application of zytase.
Detailed description of the invention
Fig. 1 is that bacterium germination Mu and mutant bacteria Mu17 colonial morphology compare figure out.
Specific embodiment
The routine techniques and method that the present invention has used genetic engineering and molecular biology field uses, such as MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT Documented method in PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These general bibliography Provide definition well known by persons skilled in the art and method.But this is not meant to limit the invention to described appoint What specific method, experimental program and reagent, because they can change.
The present invention will be described in detail With reference to embodiment.
The shake flask fermentation and Enzyme activity assay of 1 trichoderma reesei Mu of embodiment
Trichoderma reesei Mu (Trichoderma reesei Mu) strain is applicant by by the xylan in trichoderma reesei source Enzyme gene is transformed into trichoderma reesei host cell, the trichoderma reesei engineering bacteria for expressed xylanase constructed Strain.Applicant is fermented to prepare zytase always using the bacterial strain, but find in use the bacterial strain there is The problem of producing enzyme vigor reduces.Therefore, applicant carries out mutagenesis using Mu plants of trichoderma reesei as starting strain, and screening is adapted to big The horizontal high mutant strain of scale evaluation, producing enzyme.
Trichoderma reesei Mu is inoculated into fresh PDA plate (potato 200g/L, after boiling 20-30min first by applicant Filtering and removing slag；Glucose 2%；Agar powder 1.5%), 30 DEG C of culture 7d.
The sterile water elution of 5ml is drawn, spore liquid is obtained, is inoculated with 50ml MM fermentation medium (1.5% glucose, 4% liquid Sugar, 2.5% corn pulp, 0.44% (NH4)2SO4, 0.09%MgSO4, 2%KH2PO4, 0.04%CaCl2, 0.018% tween- 80,0.018% microelement, 0.018% polypropylene glycol -2000), 28 DEG C are cultivated 120 hours, and centrifugation obtains fermented supernatant fluid. Fermented supernatant fluid is subjected to the measurement of xylanase activity power.The results show that xylanase activity is in the fermented supernatant fluid 833U/mL。
Enzyme activity determination method
(1) definition of xylanase activity unit
Under conditions of 37 DEG C, pH value are 5.5,1 μm of ol is discharged from the xylan solution that concentration is 5mg/ml per minute Enzyme amount required for reduced sugar is an enzyme activity unit U.
(2) enzyme activity determination method
Taking 2ml concentration is 1% xylan substrate (preparation of pH5.5 acetic acid-sodium acetate buffer solution), is added to colorimetric cylinder In, 37 DEG C of balance 10min add the acid that 2ml is suitably diluted through pH5.5 acetic acid-sodium acetate buffer solution and balanced through 37 DEG C Property zytase enzyme solution, mix in 37 DEG C of accurate insulation reaction 30min.After reaction, be added 5ml DNS reagent, mix with Terminate reaction.Then boiling water bath boils 5min, is cooled to room temperature with tap water, and distilled water is added to be settled to 25ml, after mixing, with mark Quasi- blank sample is blank control, and light absorption value A is measured at 540nmE。
Enzyme activity calculation formula:
XD=[(AE- AB)×K+C0]×N×1000/(M×t)
In formula: XDFor the vigor of zytase in dilution enzyme solution, U/ml；AEFor the absorbance of enzyme reaction solution；ABFor enzyme blank The absorbance of liquid；K is the slope of standard curve；C0For the intercept of standard curve；M is the molal weight of xylose, 150.2g/mol； T is enzyme digestion reaction time, min；N is enzyme solution extension rate；1000 be transforming factor, 1mmol=1000 μm of ol.
2 Uv-induced screening of embodiment
Applicant further screens zytase yield by the method for ultraviolet mutagenesis using trichoderma reesei Mu as starting strain The mutant strain of raising.
It determines lethality: above-mentioned trichoderma reesei Mu is inoculated in PDA plate, 30 DEG C of culture 7d.It is generated to bacterium colony surface big When measuring spore, the sterile water elution of 5ml is drawn, spore liquid is obtained, is resuspended after centrifugation with sterile water, is counted with blood counting chamber.It takes One 90mm culture dish, the spore suspension that 5ml has diluted is added, and (concentration is about 1 × 107A/mL), rotor is added and in magnetic force Stirring makes spore liquid be in uniform state on blender.In aseptic superclean bench, the ultraviolet lamp for being 9w with power is in vertical The top of distance 20cm is irradiated, and irradiates 30s, 60s, 90s, 120s, 150s, 180s respectively, spore liquid dilution 10 after taking irradiation, 100, it 1000 times, takes 100ul to be coated with PDA plate, is counted after 30 DEG C of culture 2-3d, be control with non-irradiated spore liquid, calculate Lethality.When wherein irradiating 150s, lethality 98% chooses the irradiation time and carries out subsequent Mutagenesis experiments.
Mutagenesis screening: taking a 90mm culture dish, and the spore suspension that 5ml has diluted is added, and (concentration is 1 × 107A/mL), Rotor is added and stirring on magnetic stirring apparatus makes spore liquid be in uniform state.In aseptic superclean bench, it is with power The ultraviolet lamp of 9w is irradiated in the top of vertical range 25cm, after irradiating 150s, is closed ultraviolet lamp tube, is covered culture dish upper cover, black Dark static 20min.Then the spore suspension crossed through ultraviolet irradiation is diluted 100 times, takes 200ul to be coated with a PDA plate, often Criticize 20 PDA plates of coating, 30 DEG C of culture 2d.Morphologic observation is carried out to the bacterium colony grown on PDA plate first, picks out bacterium colony Totally 34 plants of the mutant bacteria obviously to become smaller, it is inoculated in PDA plate, 30 DEG C of culture 7d respectively.Each mutant bacteria bacterium colony 5ml sterile water It is eluted, obtains spore liquid；It is seeded to 50ml MM fermentation medium (1.5% glucose, 4% liquid sugar, 2.5% corn respectively Slurry, 0.44% (NH4)2SO4, 0.09%MgSO4, 2%KH2PO4, 0.04%CaCl2, 0.018% Tween-80,0.018% is micro Element, 0.018% polypropylene glycol -2000), 28 DEG C are cultivated 120 hours；Centrifugation obtains fermented supernatant fluid.
By carrying out the detection of xylanase activity power to the fermented supernatant fluid of above-mentioned acquisition, applicant's finishing screen selects one plant The highest mutant strain of zytase yield is named as trichoderma reesei Mu17 (Trichoderma reesei Mu17), the bacterial strain Xylanase activity reaches 1500U/mL in fermented supernatant fluid, and bacterium germination improves 80% more out, achieves unexpected technology effect Fruit.
The mutant bacteria Mu17 is compared with bacterium germination Mu out and is changed significantly (as shown in Figure 1) in bacterium colony phenotype, mutation The bacterium colony of bacterium Mu17 obviously becomes smaller, and colony diameter is only the 1/2 of bacterium germination, and bacterium colony is more closely knit, advantageously reduces zymocyte liquid Viscosity improves the dissolved oxygen content in fermentation process, and then improves biomass, increases the secreting, expressing amount of foreign protein.
Applicant is on December 15th, 2017 by mutant strain trichoderma reesei Mu17 (the Trichoderma reesei Mu17 it) is preserved in the China typical culture collection center of Wuhan, China Wuhan University, deposit number is CCTCC NO: M2017797。

Claims (6)

1. a kind of trichoderma reesei, which is characterized in that the deposit number of the trichoderma reesei is CCTCCNO:M2017797.
2. application of the trichoderma reesei described in claim 1 in production zytase.
3. a kind of method for producing zytase, which is characterized in that the method is with trichoderma reesei described in claim 1 It is fermented as fermentation strain to produce zytase.
4. method as claimed in claim 3, which is characterized in that fermentation medium used in the fermentation, composition is such as Under: 1.5% glucose, 4% liquid sugar, 2.5% corn pulp, 0.44% (NH4)2SO4, 0.09%MgSO4, 2%KH2PO4, 0.04% CaCl2, 0.018% Tween-80,0.018% microelement, 0.018% polypropylene glycol -2000.
5. a kind of zytase, which is characterized in that the zytase is obtained by trichoderma reesei described in claim 1 fermentation ?.
6. zytase as claimed in claim 5, which is characterized in that the zytase is by as claimed in claim 4 Method preparation.
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CN110564629A (en) * 2019-07-04 2019-12-13 山东百龙创园生物科技股份有限公司 trichoderma reesei and culture method and application thereof
WO2021000963A1 (en) * 2019-07-04 2021-01-07 山东百龙创园生物科技股份有限公司 Trichoderma reesei strain, culture method therefor, and application thereof

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Publication number Priority date Publication date Assignee Title
CN110564629A (en) * 2019-07-04 2019-12-13 山东百龙创园生物科技股份有限公司 trichoderma reesei and culture method and application thereof
WO2021000963A1 (en) * 2019-07-04 2021-01-07 山东百龙创园生物科技股份有限公司 Trichoderma reesei strain, culture method therefor, and application thereof

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