CN110938554B

CN110938554B - Aspergillus niger mutant strain capable of stably producing lipase at high yield

Info

Publication number: CN110938554B
Application number: CN201911179910.XA
Authority: CN
Inventors: 宋清清; 徐晓东; 田延军; 黄亦钧
Original assignee: Qingdao Master Biological Technology Co ltd; Qingdao Vland Biotech Group Co Ltd
Current assignee: Qingdao Master Biological Technology Co ltd; Qingdao Vland Biotech Group Co Ltd
Priority date: 2019-11-27
Filing date: 2019-11-27
Publication date: 2022-10-28
Anticipated expiration: 2039-11-27
Also published as: CN110938554A

Abstract

The invention belongs to the technical field of microbial engineering modification, and provides an Aspergillus niger mutant strain with high lipase yield and application thereof. The mutant strain is obtained by ultraviolet mutagenesis screening, and the preservation number is CCTCC NO: and M2019945. The strain can be widely applied to the production of lipase, thereby being beneficial to reducing the production cost of the lipase and promoting the popularization and application of the lipase in the fields of aquatic feed additives, grease processing, fine chemicals, medical intermediates and the like.

Description

Aspergillus niger mutant strain capable of stably producing lipase at high yield

Technical Field

The invention belongs to the technical field of microbial modification, and particularly relates to aspergillus niger with stable and high lipase yield and application thereof in lipase production.

Background

Lipases (EC 3.1. L.3), also known as triacylglycerol hydrolases, catalyze the hydrolysis, alcoholysis, esterification, transesterification of triacylglycerides and other water-insoluble esters and the reverse synthesis of esters, in addition to which phospholipase, lysophospholipase and cholesterol esterase activities, etc., are also present.

The catalytic property of lipase is different from that of other hydrolytic enzymes, the hydrolysis reaction is a heterogeneous system, only enzyme dissolved in water at the interface of substrate (water-insoluble) and water catalyzes the substrate reaction, but water-soluble substrate is not catalyzed, namely the catalytic activity of lipase on oil-water interface is maximum. The lipase catalytic reaction has the characteristics of stereoselectivity, substrate specificity, less side reaction, mild reaction condition, no need of coenzyme, capability of being used for organic solvents and the like, and has application in a plurality of production fields of food, leather, biodiesel, medical medicine, detergents and the like, such as food seasoning synthesis, detergent application as an additive, chiral medicine resolution, fine chemical product synthesis and the like.

As early as 100 years ago, a study of lipase was started. With the sequential reports of rabbit pancreatic lipase, gastric lipase and plant seed lipase, microbial lipase was also found at the beginning of 20. Although the research on microbial lipases starts late, the lipases are widely distributed in microorganisms, so that microbial lipases which are deeply researched have been increasing in recent 30 years and are applied to industrial production, wherein aspergillus, rhizopus, mucor, pseudomonas, geobacteria, bacillus and the like are main production bacteria. According to preliminary estimation, about 65 genera of microorganisms in nature can produce lipase, wherein 4 genera of actinomycetes, 28 genera of bacteria, 10 genera of yeast and 23 genera of other fungi exist, but actually, the distribution of lipase in microbial communities far exceeds the number. Because the microorganisms are various in types and easy to culture, the enzyme yield can be improved by a breeding means, and the microorganisms have wider action temperature, action pH and substrate specificity than animal and plant lipases, so that finding the lipase from the microbial metabolites becomes a research hotspot of researchers.

In recent years, china has made intensive studies on screening strains producing lipase by microorganisms, optimizing industrial fermentation conditions and genetic engineering. Compared with the foreign countries, the research and development of lipase in China are late, and the types of industrial microbial lipase enzyme preparations are limited, so that the screening of lipase strains with new characteristics, high activity, high yield and low cost, the improvement of the characteristics of low enzyme yield of wild strains and the development of new lipase varieties by using high-end genetic engineering technology and the like are still required to be deeply researched, the application fields of the lipase are widened, and the requirements of various related industrial fields are met.

Disclosure of Invention

The invention provides a stable aspergillus niger (a) with high lipase yield for solving the problems of the prior artAspergillus niger) And (3) strain. The applicant first derived from Candida antarctica (C.), (Candida antarctica) The lipase gene is transformed into Aspergillus niger host cells, an Aspergillus niger engineering strain for recombining and expressing the lipase is constructed, and then the lipase is mutagenized by ultravioletThe method screens and obtains a mutant strain with obviously improved lipase yield, can be applied to the production of lipase, is beneficial to reducing the production cost and promotes the wide application of the lipase.

The invention provides an Aspergillus niger engineering strain which carries a recombinant vector for expressing lipase genes.

The invention also provides a mutant strain Aspergillus niger SU9-LipBAspergillus niger SU 9-LipB), which has been preserved in the China center for type culture Collection (CCTCC NO: M2019945) of the university of Wuhan, china in 2019, 11 months and 18 days.

The invention also provides application of the Aspergillus niger mutant strain in lipase production.

After the mutant strain Aspergillus niger SU9-LipB 20L tank is fermented for 160 hours, the lipase activity in the fermented supernatant reaches 2600 LU/ml, the total protein reaches 3g/L, and the lipase activity is respectively improved by 73% and 67% compared with the original strain. The optimum action pH of the lipase expressed by the aspergillus niger mutant strain is 7.0, and the optimum action temperature is 60 ℃. The strain can be widely applied to the production of lipase, thereby being beneficial to reducing the production cost of the lipase and promoting the popularization and application of the strain in the fields of aquatic feed additives, grease processing, fine chemicals, medical intermediates and the like.

Detailed Description

The process of the present invention is further illustrated below with reference to examples. The experimental procedures, for which specific conditions are not noted in the following examples, can generally be run under conventional conditions, such as those described in molecular cloning guidelines written by J.Sambruke (Sambrook), et al, or as recommended by the manufacturer. The present invention may be better understood and appreciated by those skilled in the art with reference to the examples. However, the protection and scope of the claims is not limited to the specific examples provided, but includes the scope of protection that can be extended by a person skilled in the art without inventive work on the basis of the present description.

Example 1 Synthesis and amplification of Lipase Gene

According to the gene sequence (GeneBank Z30645.1) of lipase CALB published by NCBI, 8 bases of TCTAGAGC (XbaI restriction site shown by underlining) are added before the initiation codon ATG of the lipase CALB, 9 bases of CCGCTCGAG (XhoI restriction site shown by underlining) are added after the termination codon TGA of the lipase CALB, then the sequence is subjected to codon optimization according to the codon preference of Aspergillus niger, and the optimized gene sequence is synthesized by Nanjing King Kingsley Biotech Limited.

The lipase gene is cut by restriction enzymes XbaI and XhoI (Fermentas); meanwhile, the plasmid pS2 was digested with restriction enzymes XbaI and XhoI. The cleavage products were purified using a gel purification kit and ligated with T4 DNA ligase (Fermentas). The ligation product was transformed into DH 5. Alpha. E.coli and selected with ampicillin. To ensure accuracy, several clones were sequenced (Invitrogen).

Plasmids were purified from E.coli clones with correct sequencing using a plasmid miniprep kit (Axygen). 1 recombinant plasmid pS2-lipB was obtained.

EXAMPLE 2 construction of Aspergillus niger engineering strains

1. Preparing protoplasts:

sucking an Aspergillus niger host SU9 spore suspension liquid on a PDA (personal digital assistant) plate, cutting the culture medium with the size of 2cm multiplied by 2cm into 200 mL of CMA liquid culture medium when bacterial colonies grow on the whole culture dish, and culturing for 14 to 16 hours at the temperature of 30 ℃ and the rpm of 200.

Collecting mycelium with sterile Miracloth filter cloth, washing the mycelium with the solution A once, transferring the washed mycelium into 40 mL of protoplast transformation solution under the sterile condition, carrying out warm bath for 2-3 h under the conditions of 30 ℃ and 200 rpm, and observing the protoplast under a microscope to ensure complete lysis.

And (3) filtering the warm bath liquid by using a sterile Miracloth filter cloth to obtain a filtrate, namely the protoplast solution. The protoplast solution was dispensed into two 50mL disposable sterile centrifuge tubes and the volume of each tube was brought to 45 mL with solution B, centrifuged at 3000 rpm for 10 min to obtain a pellet and the supernatant discarded. The pellet was resuspended and washed twice with 20 mL of solution B. Resuspend pellet in 10 mL of solution B and use bloodThe protoplasts were counted on a ball counter. Centrifuging the protoplast again and discarding supernatant, adding appropriate amount of solution B to resuspend the precipitate according to counting result of blood counting plate to make the number of protoplast at 1 × 10 ⁷ one/mL.

2. And (3) transformation:

200. Mu.L of the above protoplast solution was added to a pre-cooled sterile 15 mL centrifuge tube on ice, 1 tube for each transformation reaction, 10. Mu.g of recombinant plasmid pS2-lipB and 50. Mu.L of solution C were added, mixed gently, and then placed on ice for 20 min. The MMSA top-layer agarose was melted and kept at 55 ℃. And removing the 15 mL centrifuge tube from the ice, adding 2 mL of the solution C into the tube, standing at room temperature for 5min, adding 4 mL of the solution B, and gently mixing the tubes to obtain a mixture, namely the protoplast mixture. And adding the protoplast mixture into a top agar culture medium, immediately mixing uniformly, pouring the mixture onto an MMSA (MMSA) plate, standing at room temperature for solidification, and culturing the plate at 30 ℃ for 7-10 d.

Solution A:2.5 mL 1M K ₂ HPO4，2.5 mL 1M KH ₂ PO4, 48.156 g MgSO4, adding ddH ₂ O to a final volume of 500 mL, and filter sterilized with a 0.22 μm microporous filter.

Solution B: 5mL 1M Tris (pH 7.5), 2.77 g CaCl ₂ 109.32 g sorbitol, ddH was added ₂ O to a final volume of 500 mL, and filter sterilized with a 0.22 μm microporous filter.

Solution C:250 g PEG 4000,2.77 g CaCl ₂ 5mL 1M Tris (pH 7.5), ddH was added ₂ O to a final volume of 500 mL, and filter sterilized with a 0.22 μm microporous filter.

MMSA culture medium: 20 glucose (g), malt extract (20 g), peptone (1 g), agar (15 g), ddH ₂ O to a final volume of 1000 mL, and autoclaving.

3. And (3) transformant screening:

after culturing for 5 days, selecting grown colonies, dibbling the colonies to a transformation lower layer plate for rescreening, and culturing for 5 days at the temperature of 30 ℃. The transformants which grew normally were inoculated into fresh PDA plates, respectively, and cultured at 30 ℃ for 5-7 days. Each transformant was harvested into 2cm × 2cm pieces, inoculated into 50ml of TSB fermentation medium, fermented at 30 deg.C for 5 days. After the culture is finished, the thalli are centrifuged to obtain supernatant, and SDS-PAGE protein electrophoresis detection and lipase enzyme activity detection are respectively carried out.

Electrophoresis results show that the supernatant fluid of the aspergillus niger recombinant strain fermentation obtained by the construction method has target protein bands at the 33kD position, and the target protein bands are consistent with the theoretical molecular weight of the lipase LipB, so that the recombinant strain obtained by the construction method can effectively express the lipase LipB. The applicant named Aspergillus niger SU9-lipB from a recombinant strain with highest lipase activity in fermentation supernatantAspergillus niger SU 9-lipB), wherein the lipase activity in the fermentation supernatant is 100 LU/mL.

Method for measuring lipase activity

Detecting the enzyme activity of Aspergillus niger fermentation liquid according to the method of GB/T23535 lipase preparation, wherein the enzyme activity is defined as follows: 1g of solid enzyme powder (or 1mL of liquid enzyme), hydrolyzing the tributyrin for 1min at 30 ℃ and pH8.0 to generate 1 mu mol of titratable fatty acid, namely an enzyme activity unit expressed by LU/g (LU/mL).

Example 3 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 Aspergillus niger SU9-lipB as an original strain, and performs genetic modification on the Aspergillus niger SU9-lipB by an ultraviolet mutagenesis method, so as to further improve the yield of lipase.

1. Determination of the lethality rate:

aspergillus niger SU9-lipB was inoculated to PDA plates and cultured at 30 ℃ for 5-7 days. When a large amount of spores are generated on the surfaces of the colonies, 5ml of sterile water is absorbed for elution to obtain a spore solution, the spore solution is re-suspended by sterile water after centrifugation, and the spore solution is counted by a blood counting chamber. Taking a 90mm culture dish, adding 5ml of diluentReleased spore suspension (concentration 1X 10) ⁷ one/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 ultra-clean bench for 30s, 45s, 60s, 75s, 90s, 105s and 120s, respectively, diluting the irradiated spore solution by 100 times and 1000 times, coating 100ul PDA plate with the diluted spore solution, culturing at 30 deg.C for 2-3d, counting, and calculating lethality with unirradiated spore solution as control. Wherein the lethality was 95% at 60s irradiation time, which was selected for subsequent mutagenesis experiments.

2. First round mutagenesis screening:

a90 mm petri dish was taken and 5ml of diluted spore suspension (concentration 1X 10) was added ⁷ one/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 above 20cm in sterile ultra-clean bench, diluting 1000 times after irradiating for 60s, taking 100ul PDA coated plate, and culturing at 30 deg.C for 2-3d.

150 PDA plates are coated in total, 40-60 colonies grow on each plate after being cultured at 30 ℃ for 2-3d, and mutants with short branches are screened out through colony morphology. The applicant picks out 120 mutant bacteria with small colony morphology, compact hyphae and short villus around the colony, and the mutant bacteria are respectively inoculated to a PDA plate and cultured for 5-7 days at 30 ℃. Each transformant was harvested by cutting 2cm × 2 cm-sized clumps of bacteria, inoculated into 50ml of liquid shake flask medium, fermented, and cultured at 30 ℃ for 5 days. After the culture is finished, centrifuging the thalli to obtain supernatant fluid, namely crude enzyme liquid, respectively carrying out lipase activity detection, and simultaneously taking an original strain Aspergillus niger engineering bacterium SU9-lipB as a control group.

The result shows that the enzyme activity of lipase in the fermentation supernatant of no mutant strain is higher than that of the original strain in 120 mutant strains obtained by the first round of ultraviolet mutagenesis screening; wherein, the enzyme activity of 86 mutant strains is basically equivalent to that of the original strain, and the enzyme activity of the rest 34 mutant strains is reduced by 10-20 percent compared with that of the original strain.

The applicant continues to carry out 8 rounds of mutagenesis screening according to the method, and finally obtains 1 mutant strain with lipase yield remarkably higher than that of the developed strainNamed as Aspergillus niger SU 9-LipB: (Aspergillus niger SU 9-LipB). The enzyme activity of lipase in the Aspergillus niger SU9-LipB shake flask fermentation supernatant reaches 225 LU/ml, which is 56% higher than that of the original strain.

Further, the applicant fermented the starting strain Aspergillus niger SU9-lipB and the mutant strain Aspergillus niger SU9-lipB in 20L tanks, respectively. And after fermenting for 160h, centrifuging the thalli to obtain supernatant, namely crude enzyme liquid, and respectively carrying out lipase activity detection.

The results show that the lipase activity in the supernatant fluid fermented by the original strain Aspergillus niger SU9-lipB is 1500 LU/ml, and the protein amount is 1.8g/L, while the lipase activity in the supernatant fluid fermented by the mutant strain Aspergillus niger SU9-lipB is as high as 2600 LU/ml, and the protein amount is 3g/L, which are respectively improved by 73% and 67% compared with the original strain, and unexpected technical effects are achieved.

The mutant strain Aspergillus niger SU9-LipB (I.A.)Aspergillus niger SU 9-LipB) is preserved in China center for type culture Collection (CCTCC NO: M2019945) of the university of Wuhan, china.

Example 4 analysis of enzymatic Properties

1. Optimum pH for action

Respectively preparing phosphate buffer solutions with pH values of 5.0, 6.5, 7.0, 8.0 and 9.0, respectively diluting fermentation supernatant of mutant bacteria Aspergillus niger SU9-LipB to appropriate concentrations by using the buffer solutions, and then measuring the enzyme activity of lipase in the fermentation supernatant according to the method of GB/T23535. Calculating the relative enzyme activity of the lipase by taking the original enzyme activity as 100 percent. The result shows that the optimum action pH of the lipase expressed by the Aspergillus niger SU9-LipB recombination is 7.0.

2. Optimum temperature of action

Diluting the fermentation supernatant of the mutant strain Aspergillus niger SU9-LipB to a proper concentration by the buffer solution with the pH of 7.0, and measuring the enzyme activity of lipase in the fermentation supernatant according to the method of GB/T23535 at the conditions of 30 ℃, 40 ℃,50 ℃, 60 ℃, 65 ℃ and 70 ℃. The relative enzyme activity of the lipase under different temperature conditions is calculated by taking the original enzyme activity as 100 percent. The result shows that the optimal action temperature of the lipase expressed by the Aspergillus niger SU9-LipB recombination is 60 ℃.

The lipase can be widely applied to the fields of aquatic feed additives, grease processing, fine chemicals, medical intermediates and the like.

Claims

1. A new kind of Aspergillus nigerAspergillus niger) The mutant is characterized in that the preservation number of the mutant is CCTCC NO: M2019945.

2. Use of the mutant of claim 1 for producing lipase.