CN111349569B - Trichoderma reesei and application thereof in xylanase production - Google Patents
Trichoderma reesei and application thereof in xylanase production Download PDFInfo
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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
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- C12N9/14—Hydrolases (3)
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- 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
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- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
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Abstract
The invention belongs to the technical field of microorganism modification and screening, and particularly relates to a trichoderma reesei mutant strain and application thereof in xylanase production. The preservation number of the mutant strain is CCTCC NO: M2018885. After the mutant strain is subjected to shake flask fermentation for 120 hours, the xylanase activity in the fermentation supernatant reaches 1980U/mL, which is 32% higher than that of the original strain; after fermentation is carried out for 160h in a 20L tank, the xylanase activity in the fermentation supernatant reaches 32050U/mL, is improved by 68 percent compared with the original strain, and obtains unexpected technical effect. The Trichoderma reesei mutant strain provided by the invention can be widely applied to fermentation production of xylanase, is beneficial to reducing the production cost of the xylanase and promotes the popularization and application of the xylanase.
Description
Technical Field
The invention belongs to the technical field of microorganism modification and screening, and particularly relates to a Trichoderma reesei strain and application thereof in xylanase production.
Background
Xylanase refers to an enzyme capable of degrading beta-1, 4-xylan to produce xylose, and the enzyme can degrade hemicellulose which is a main component of cell walls. Therefore, the enzyme plays a very important role in the process of generating available nutrients by degrading plant cell walls through microorganisms. Xylanases are commonly produced by fungi, bacteria, yeast, algae, and the like. Commercial xylanases are produced primarily by fermentation of filamentous fungi. At present, the application of xylanase in feed is mature day by day, and the xylanase is widely applied to the feed of livestock and poultry.
The energy feed in China mainly comprises corns, wheat, grains, bran and the like, and the arabinoxylan is one of the main components in the daily ration and cannot be digested and utilized by monogastric animals such as poultry and the like. In the case of wheat, approximately 114g/kg of non-starchy polysaccharides (NSP) are present, these being mainly arabinoxylans. Approximately 210g/kg of non-starchy polysaccharide (NSP) is water soluble. Arabinoxylan has shown an anti-nutritional effect in monogastric animals. The water-soluble arabinoxylans form highly viscous liquids, which hinder the contact of digestive enzymes and nutrient substrates, resulting in a hindered nutrient uptake. Insoluble non-starchy polysaccharides physically obstruct the binding of enzymes to nutrient substrates. The digestive utilization rate of the feed in animal bodies can be well improved by adding other enzyme preparations such as xylanase and the like into the feed.
With the continuous development of genetic engineering technology, xylanase genes can be expressed in expression systems of escherichia coli, bacillus, yeast and filamentous fungi. For example, XynA gene is cloned from the schizophyllum thermophilum and expressed in pichia pastoris X-33, the recombinase protein can maintain 60 percent of enzyme activity in the environment with pH6.0-9.0 and 60-80 ℃, and the enzyme has larger application potential due to higher tolerance to pH and temperature. The xylanase gene Xyn11A cloned from bacillus encodes 366 amino acids, and the optimal reaction temperature of the recombinant protease is 55 ℃. The XynB gene of the Aspergillus niger IA-001 is optimized and cloned into Pichia pastoris GS115, the activity of the optimized recombinant protein is improved by 2.8 times compared with that of a wild enzyme, and the recombinant protein can express higher xylanase activity.
The production strain with improved xylanase activity is obtained by screening through a mutagenesis method, so that the method is suitable for industrial large-scale production and promotes the further development of xylanase.
Disclosure of Invention
The invention aims to provide a Trichoderma reesei (Trichoderma reesei) strain and application thereof in xylanase production. The mutant strain with greatly improved xylanase yield is obtained by screening through an ultraviolet mutagenesis method, can be widely applied to the production of xylanase, reduces the cost of the xylanase, and is beneficial to the wide application of the xylanase.
The invention provides a mutant strain Trichoderma reesei SC-2F64(Trichoderma reesei SC-2F64) which is preserved in China center for type culture Collection of Wuhan university in Wuhan, China in 12 months and 12 days in 2018, wherein the preservation number is CCTCC NO: M2018885.
In one aspect, the invention provides an application of the trichoderma reesei in xylanase production.
The invention also provides a method for producing xylanase, which takes the trichoderma reesei as a fermentation strain.
The invention also provides xylanase obtained by fermenting the trichoderma reesei.
The invention takes Trichoderma reesei Mu17 as an original strain, and obtains a mutant strain Trichoderma reesei SC-2F64 by screening through an ultraviolet mutagenesis method. After the mutant strain is subjected to shake flask fermentation for 120 hours, the xylanase activity in the fermentation supernatant reaches 1980U/mL, which is 32% higher than that of the original strain; after fermentation is carried out for 160h in a 20L tank, the xylanase activity in the fermentation supernatant reaches 32050U/mL, is improved by 68 percent compared with the original strain, and obtains unexpected technical effect. The Trichoderma reesei mutant strain provided by the invention can be widely applied to fermentation production of xylanase, is beneficial to reducing the production cost of the xylanase and promotes the popularization and application of the xylanase.
Detailed Description
The invention uses the conventional technology and method for modifying and screening microorganisms. Those skilled in the art can adopt other conventional methods, experimental schemes and reagents based on the technical scheme described in the invention, and the invention is not limited to the specific embodiment of the invention.
The present invention will be described in detail with reference to specific embodiments.
Example 1 Shake flask fermentation and enzyme Activity detection of Trichoderma reesei Mu17
The applicant constructs a Trichoderma reesei engineering strain mu (Trichoderma reesei mu) for recombining and expressing xylanase by transforming xylanase genes derived from Trichoderma reesei into Trichoderma reesei host cells. In order to improve the xylanase yield of the Trichoderma reesei Mu, the applicant takes the engineering bacteria as starting bacteria and obtains a mutant strain Trichoderma reesei Mu17(Trichoderma reesei Mu17) by screening through an ultraviolet mutagenesis method. The xylanase yield of the mutant strain is improved by 80 percent compared with that of the original strain. The mutant Trichoderma reesei Mu17(Trichoderma reesei Mu17) was deposited in China center for type culture Collection, CCTCC NO: M2017797, 12, 15, 2017, at Wuhan university, Wuhan, China. (the contents of this part are described in detail in the Chinese patent application No. 201711477714.1)
Inoculating Trichoderma reesei Mu17 to fresh PDA plate (potato 200g/L, boiling for 20-30min, filtering to remove residue, glucose 2%, agar powder 1.5%), and culturing at 30 deg.C for 7 d.
Eluting with 5ml sterile water to obtain spore liquid, inoculating 50ml MM fermentation medium (1.5% glucose, 4% liquid sugar, 2.5% corn steep liquor, 0.44% (NH)4)2SO4，0.09％MgSO4，2％KH2PO4，0.04％CaCl20.018% Tween-80, 0.018% microelement and 0.018% polypropylene glycol-2000), culturing at 28 deg.C for 120 hr, and centrifuging to obtain fermentation supernatant. And (4) carrying out enzyme activity determination on the fermentation supernatant. The result shows that the xylanase activity in the fermentation supernatant is 1500U/mL.
Enzyme activity measuring method
(1) Definition of xylanase Activity Unit
The enzyme amount required for releasing 1 mu mol of reducing sugar from 5mg/ml xylan solution per minute at 37 ℃ and pH5.5 is an enzyme activity unit U.
(2) Enzyme activity determination method
Taking 2ml of xylan substrate with the concentration of 1% (prepared by a pH5.5 acetic acid-sodium acetate buffer solution), adding the xylan substrate into a colorimetric tube, balancing for 10min at 37 ℃, adding 2ml of acidic xylanase enzyme solution which is properly diluted by the pH5.5 acetic acid-sodium acetate buffer solution and well balanced at 37 ℃, uniformly mixing, and accurately preserving the temperature at 37 ℃ for reaction for 30 min. After the reaction was completed, 5ml of DNS reagent was added and mixed well to terminate the reaction. Boiling in boiling water bath for 5min, cooling to room temperature with tap water, adding distilled water to desired volume of 25ml, mixing, and making into blank sample at 540nmDetermination of the Absorbance AE。
The enzyme activity calculation formula is as follows:
XD＝[(AE－AB)×K+C0]×N×1000/(M×t)
in the formula: xDFor the activity of xylanase in the diluted enzyme solution, U/ml; a. theEThe absorbance of the enzyme reaction solution; a. theBThe absorbance of the enzyme blank liquid; k is the slope of the standard curve; c0Is the intercept of the standard curve; m is the molar mass of xylose, 150.2 g/mol; t is enzymolysis reaction time, min; n is the dilution multiple of enzyme solution; 1000 is conversion factor, 1mmol ═ 1000 μmol.
Example 2 UV mutagenesis screening
The mutation caused by ultraviolet mutagenesis has strong randomness, and the effect generated by mutation is random and difficult to predict. Therefore, in order to obtain effective positive mutations, technicians usually need to perform multiple rounds of ultraviolet mutagenesis, the screening workload is large, and the possibility that effective positive mutations cannot be obtained exists. However, ultraviolet mutagenesis requires simple equipment and low cost, and can obtain a large number of mutants in a short time, so that it is still a common mutagenesis breeding method.
The applicant takes trichoderma reesei Mu17 as an original strain, and carries out genetic modification on the trichoderma reesei Mu17 by an ultraviolet mutagenesis method, so that the yield of xylanase is further improved.
2.1 determination of lethality
The above Trichoderma reesei Mu17 was inoculated on a PDA plate and cultured at 30 ℃ for 7 days. When a large amount of spores are generated on the surface of the colony, 5ml of sterile water is absorbed for elution to obtain a spore liquid, the spore liquid is resuspended by the sterile water after centrifugation, and a blood counting chamber is used for counting. A90 mm petri dish was taken and 5ml of diluted spore suspension (concentration about 1X 10) was added7one/mL) was added to the vessel and stirred on a magnetic stirrer to make the spore liquid homogeneous. Irradiating with ultraviolet lamp with power of 9w at a vertical distance of 20cm in a sterile ultraclean bench for 30s, 60s, 90s, 120s, 150s, and 180s, respectively, diluting the irradiated spore solution for 10, 100, and 1000 times, coating 100ul PDA plate with the diluted spore solution, culturing at 30 deg.C for 2-3d, counting, and countingSpore liquid is used as a control, and the lethality rate is calculated. Wherein the lethality is 98% when the irradiation time is 150s, and the irradiation time is selected for subsequent mutagenesis experiments.
2.2 first round mutagenesis screening
A90 mm petri dish was taken and 5ml of diluted spore suspension (concentration 1X 10) was added7one/mL) was added to the vessel and stirred on a magnetic stirrer to make the spore liquid homogeneous. Irradiating with ultraviolet lamp with power of 9w at a vertical distance of 25cm in a sterile ultra-clean workbench for 150s, closing the ultraviolet lamp tube, covering the upper cover of the culture dish, and standing in dark for 20 min. The UV-irradiated spore suspension was then diluted 100-fold, 200ul of the suspension was plated on a PDA plate, 20 PDA plates were plated in each batch, and the batch was incubated at 30 ℃ for 2 days. Firstly, morphologically observing colonies growing on a PDA plate, selecting 58 mutant bacteria with obviously reduced colonies, respectively inoculating the mutant bacteria on the PDA plate, and culturing at 30 ℃ for 7 d. Eluting each mutant bacterium colony by using 5ml of sterile water to obtain spore liquid; separately, 50ml of MM fermentation medium (1.5% glucose, 4% liquid sugar, 2.5% corn steep liquor, 0.44% (NH)4)2SO4，0.09％MgSO4，2％KH2PO4，0.04％CaCl20.018% Tween-80, 0.018% microelement and 0.018% polypropylene glycol-2000), and culturing at 28 deg.C for 120 hr; centrifuging to obtain fermentation supernatant, respectively measuring the xylanase activity, and simultaneously taking the original strain trichoderma reesei Mu17 as a control group.
The result shows that the enzyme activity of xylanase in the supernatant obtained by fermentation of no mutant strain is higher than that of the original strain in 58 mutant strains obtained by the first round of ultraviolet mutagenesis screening.
The applicant carries out 11 rounds of mutagenesis screening according to the method, and finally obtains 1 mutant strain with xylanase yield remarkably higher than that of the original strain, namely trichoderma reesei SC-2F 64. After the trichoderma reesei SC-2F64 is subjected to shake flask fermentation for 120h, the enzyme activity of xylanase in a fermentation supernatant is the highest and reaches 1980U/ml, the enzyme activity is improved by 32% compared with that of the original strain, and an unexpected technical effect is achieved.
The applicant further fermented the starting strain Trichoderma reesei Mu17 and the mutant strain Trichoderma reesei SC-2F64 in 20L tanks, respectively. And when the fermentation is finished for 160h, respectively measuring the enzyme activity of the xylanase in the fermentation supernatant. The result shows that the xylanase activity in the supernatant obtained by fermenting the mutant strain trichoderma reesei SC-2F64 is as high as 32050U/mL, is improved by 68 percent compared with the original strain, and obtains unexpected technical effect.
The mutant strain Trichoderma reesei SC-2F64(Trichoderma reesei SC-2F64) is preserved in China center for type culture Collection, CCTCC NO: M2018885, 12.12.2018.
Claims (3)
1. Trichoderma reesei (F.) (A.) (B.) (A. reesei)Trichoderma reesei) The preservation number of the trichoderma reesei is M2018885, and the preservation number of the trichoderma reesei is CCTCC NO.
2. Use of the trichoderma reesei of claim 1 for the production of xylanase.
3. A method for producing xylanase by fermentation of Trichoderma reesei according to claim 1 as a fermentation strain.
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