阅读说明：本技术 一种脂肪酶高产菌株及其应用 (Lipase high-yield strain and application thereof ) 是由 宋清清 李�瑞 黄亦钧 张金祥 于 2019-11-27 设计创作，主要内容包括：本发明属于微生物工程改造技术领域,提供了一种高产脂肪酶的里氏木霉突变菌株及其应用。所述突变菌株是通过紫外诱变筛选获得的,保藏编号为CCTCC NO：M2019944。所述菌株可广泛应用于脂肪酶的生产,从而有利于降低脂肪酶的生产成本,促进其在水产饲料添加剂、油脂加工、精细化学品、医药中间体等领域中的推广与应用。(The invention belongs to the technical field of microbial engineering modification, and provides a Trichoderma reesei 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: m2019944. 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.)

1. A recombinant plasmid carrying a lipase gene.

2. An engineered strain of trichoderma reesei, wherein said engineered strain carries the recombinant plasmid of claim 1.

3. The mutant strain of trichoderma reesei is characterized in that the mutant strain is obtained by taking the engineering strain of claim 2 as a starting strain and adopting an ultraviolet mutagenesis method.

4. The mutant strain of claim 3, which has a collection number of CCTCC NO of M2019944.

5. Use of the mutant of claim 4 for producing lipase.

Technical Field

The invention belongs to the technical field of microbial modification, and particularly relates to trichoderma reesei for stabilizing high-efficiency lipase and application thereof in lipase production.

Background

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

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 over 100 years ago, the study of lipases began. 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 produce lipase, among which 4 genera of actinomycetes, 28 genera of bacteria, 10 genera of yeast, and 23 genera of other fungi, but in fact, the distribution of lipase in microbial communities has far exceeded this figure. 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 industrialized 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 to widen the application field and meet the requirements of various related industrial fields.

Disclosure of Invention

The invention provides a stable and high-yield Trichoderma reesei (Trichoderma reesei) for solving the problems of the prior artTrichoderma reesei) And (3) strain. Applicant first obtained a source from Candida antarctica: (Candida antarctica) The lipase gene is transformed into a trichoderma reesei host cell to construct a trichoderma reesei engineering strain for recombinant expression of lipase, and then a mutant strain with remarkably improved lipase yield is obtained by screening through an ultraviolet mutagenesis method.

In one aspect, the invention provides a recombinant plasmid carrying a lipase gene.

The invention provides a trichoderma reesei engineering strain which carries the recombinant plasmid.

The invention also provides a mutant strain Trichoderma reesei UE-CALB (C. vulgaris)Trichoderma reeseiUE-CALB), which has been preserved in the China center for type culture Collection of Wuhan university in Wuhan, China at 11/18/2019 with the preservation number of CCTCC NO: M2019944.

The invention also provides application of the Trichoderma reesei mutant strain in producing lipase.

After the mutant strain Trichoderma reesei UE-CALB 20L tank is fermented for 160h, the lipase activity in the fermented supernatant reaches 7500 LU/ml, the total protein amount reaches 11g/L, and the lipase activity is respectively improved by 150% and 80.3% compared with the original strain. The optimum action pH of the lipase expressed by the trichoderma reesei mutant strain in a recombination way is 8.0, and the optimum action temperature is 45 ℃. 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 following examples. However, the protection of the invention and the scope of the claims are not limited to the specific cases provided, but should include the protection that can be extended by a person skilled in the art without inventive work on the basis of the present description.