阅读说明：本技术 地衣芽胞杆菌谷氨酸脱氢酶突变体S277W在聚γ-谷氨酸合成中的应用 (Application of bacillus licheniformis glutamate dehydrogenase mutant S277W in poly-gamma-glutamic acid synthesis ) 是由 陈守文 蔡冬波 杨帆 张清 马昕 陈建刚 于 2019-11-14 设计创作，主要内容包括：本发明属于基因工程和酶工程技术领域,公开了地衣芽胞杆菌谷氨酸脱氢酶突变体S277W在聚γ-谷氨酸合成中的应用。本发明通过基因组上定点突变的方式,将来源于地衣芽胞杆菌WX-02(<I>Baclicus lincheniformis</I> WX-02,中谷氨酸脱氢酶RocG的第277位丝氨酸突变为色氨酸,显著提高了谷氨酸脱氢酶催化活力,解决了目前聚γ-谷氨酸合成过程中谷氨酸供给不足的问题,突变菌株聚γ-谷氨酸产量相较于对照菌株至少提高了12%以上。本发明为聚γ-谷氨酸的高效生产提供了一种新策略。(The invention belongs to the technical field of genetic engineering and enzyme engineering, and discloses an application of a bacillus licheniformis glutamate dehydrogenase mutant S277W in poly-gamma-glutamic acid synthesis. The invention uses the mode of site-directed mutagenesis on genome to lead the bacillus licheniformis WX-02 (B) Baclicus lincheniformis WX-02, the 277 th serine of the middle glutamate dehydrogenase RocG is mutated into tryptophan, the catalytic activity of the glutamate dehydrogenase is obviously improved, the problem of insufficient supply of glutamate in the existing synthesis process of poly-gamma-glutamic acid is solved, and the yield of the mutant strain poly-gamma-glutamic acid is at least improved by over 12 percent compared with that of a control strain. The invention provides a new strategy for the efficient production of poly-gamma-glutamic acid.)

1. The application of the glutamate dehydrogenase mutant S277W in the production of poly-gamma-glutamic acid by biological fermentation is disclosed, wherein the amino acid sequence of the mutant S277W is shown in SEQ ID NO. 1.

2. The use according to claim 1, wherein the nucleotide sequence encoding the amino acid sequence shown in SEQ ID No.1 is shown in SEQ ID No. 2.

3. The use of claim 1, wherein the Bacillus licheniformis is a Bacillus licheniformis capable of producing poly-gamma-glutamic acid.

4. The use of claim 1, wherein the Bacillus licheniformis is Bacillus licheniformis (Bnfillus licheniformis: (B) (B))Bacilluslicheniformis) WX-02。

5. The use according to claim 1, wherein the fermentation medium used in the fermentation is formulated as follows:

30-90g/L glucose, 0 ~ 30g/L sodium glutamate, 0 ~ 10g/L sodium citrate, NaNO₃ 0~10 g/L，NH₄Cl10g/L，K₂HPO₄·3H₂O 1 g/L，MgSO₄·7H₂O 1 g/L，ZnSO₄·7H₂O 1 g/L，MnSO₄·H₂O 0-0.15 g/L，CaCl₂ 1 g/L；

Or 20-40g/L of glycerin, 30g/L of sodium glutamate, 10g/L of sodium citrate and NaNO₃ 10 g/L，NH₄Cl 10 g/L，K₂HPO₄·3H₂O 1 g/L，MgSO₄·7H₂O 1 g/L，ZnSO₄·7H₂O 1 g/L，MnSO₄·H₂O 0.15 g/L，CaCl₂ 1g/L。

Technical Field

The invention belongs to the technical field of enzyme engineering and genetic engineering, and particularly relates to an application of a bacillus licheniformis glutamate dehydrogenase mutant S277W in poly-gamma-glutamic acid synthesis.

Background

Poly-gamma-glutamic acid is an anionic polypeptide consisting of D/L-type glutamic acid residues linked by an amide bond between an alpha-amino group and a gamma-carboxylic acid group. It has many excellent properties due to its biological structural characteristics. The poly-gamma-glutamic acid is used as a water-soluble, biodegradable, biocompatible, edible and nontoxic biodegradable material, and can be widely applied to the fields of food, agriculture, medicines, cosmetics, environmental protection and the like. Therefore, the poly-gamma-glutamic acid has wide application prospect.

Currently, the commercial production of poly-gamma-glutamic acid mainly depends on a microbial fermentation method, but the conversion rate of glucose into poly-gamma-glutamic acid is low due to the addition of poly-gamma-glutamic acid synthesis precursors and excessive fermentation byproducts. From the current reports, the commercial production strains of poly-gamma-glutamic acid are almost completely dependent on bacillus, such as bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis and the like. These poly-gamma-glutamic acid-producing strains can be classified into L-glutamic acid-dependent and L-glutamic acid-independent strains according to nutritional requirements. L-glutamic acid-dependent strains increase production costs in commercial production. The L-glutamic acid-independent strain is a potential cell factory with low production cost, but its productivity is extremely limited. Glutamate dehydrogenase is a key enzyme in the synthesis pathway of poly-gamma-glutamate and is responsible for catalyzing alpha-ketoglutarate to form glutamate, and then the final product poly-gamma-glutamate is generated through further reaction of poly-gamma-glutamate synthetase. Because the synthesis and accumulation of intracellular glutamic acid are necessary conditions for the efficient synthesis of poly-gamma-glutamic acid, glutamate dehydrogenase is also a key enzyme for the biosynthesis of poly-gamma-glutamic acid.

Glutamate dehydrogenase, RocG, from bacillus licheniformis is a class of coenzyme-dependent dehydrogenases that catalyze the interconversion between alpha-ketoglutarate and glutamate. At present, the protein structure and the catalytic property of bacillus licheniformis glutamate dehydrogenase RocG are not researched and analyzed, and the catalytic core region is not analyzed and clarified, so that the bacillus licheniformis glutamate dehydrogenase RocG cannot be correspondingly modified. Therefore, the influence of glutamate dehydrogenase site modification on its catalytic properties and poly-gamma-glutamate biosynthesis is also unknown.

Disclosure of Invention

The invention aims to provide application of a bacillus licheniformis glutamate dehydrogenase mutant S277W poly-gamma-glutamic acid in synthesis, wherein the amino acid sequence of the glutamate dehydrogenase mutant S277W is shown in SEQ ID No. 1.

In order to achieve the purpose, the invention adopts the following technical measures:

the application of the glutamate dehydrogenase mutant S277W in the biological fermentation production of poly-gamma-glutamic acid comprises the steps of transforming the gene for coding the mutant protein into bacillus licheniformis and producing the poly-gamma-glutamic acid through fermentation, wherein the amino acid sequence of the mutant S277W is shown in SEQ ID NO. 1.

In the above-mentioned application, preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO. 2.

In the above application, preferably, the bacillus licheniformis is bacillus licheniformis capable of producing poly-gamma-glutamic acid.

In the above application, preferably, the bacillus licheniformis is bacillus licheniformis (bacillus licheniformis) WX-02.

In the above application, in the application process, the formula of the fermentation medium used in the fermentation is as follows:

30-90g/L glucose, 0-30 g/L sodium glutamate, 0-10 g/L sodium citrate and NaNO₃ 0～10g/L，NH₄Cl 10g/L，K₂HPO₄·3H₂O 1g/L，MgSO₄·7H₂O 1g/L，ZnSO₄·7H₂O 1g/L，MnSO₄·H₂O 0-0.15g/L，CaCl₂ 1g/L；

Or 20-40g/L of glycerin, 30g/L of sodium glutamate, 10g/L of sodium citrate and NaNO₃ 10g/L，NH₄Cl 10g/L，K₂HPO₄·3H₂O 1g/L，MgSO₄·7H₂O 1g/L，ZnSO₄·7H₂O 1g/L，MnSO₄·H₂O 0.15g/L，CaCl₂ 1g/L。

Compared with the prior art, the invention has the following advantages:

according to the invention, through a genome mutation mode, the 277 th serine of the glutamate dehydrogenase is mutated into tryptophan (named as mutant S277W), the catalytic activity of the glutamate dehydrogenase is obviously improved, the problem of insufficient supply of glutamic acid in the existing poly-gamma-glutamic acid synthesis process is solved, the modified strain synthesizes poly-gamma-glutamic acid, and the synthesis level of the poly-gamma-glutamic acid is improved by at least 12% compared with that of a control strain.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof. The technical scheme of the invention is a conventional scheme in the field if not specifically stated; the reagents or materials, if not specifically mentioned, are commercially available.

Test materials and reagents

1. The strain is as follows: bacillus licheniformis (Bacillus licheniformis) WX-02 with a preservation number of CCTCC NO. M208065, and strain E.coli DH5 alpha, which is purchased from Beijing Quanyu gold biotechnology Limited.

2. Enzymes and other biochemical reagents: high fidelity Taq enzyme was purchased from Wuhan Pongzi Biotechnology Ltd. The bacterial genome DNA extraction kit is purchased from Tiangen company, molecular biological reagents such as T4 DNA ligase, restriction endonuclease and the like are purchased from Nanjing Novophilia Biotech Co., Ltd, and other reagents are all made in China (all can be purchased from common biochemical reagents).

3. Culture medium:

(1) the LB culture medium formula is: 10g/L tryptone, 5g/L yeast powder, 10g/L sodium chloride, pH 7.0-7.2, sterilizing at 121 ℃ for 20min and using.

(2) Fermentation medium: 30-90g/L glucose, 0-30 g/L sodium glutamate, 0-10 g/L sodium citrate and NaNO₃0～10g/L，NH₄Cl 0～5g/L，K₂HPO₄·3H₂O 0～1g/L，MgSO₄·7H₂O 0～1g/L，ZnSO₄·7H₂O 0～1g/L，MnSO₄·H₂O 0～0.15g/L，CaCl₂ 0.5～1g/L；

Or: 20-60g/L of glycerin, 30g/L of sodium glutamate, 10g/L of sodium citrate and NaNO₃ 10g/L，NH₄Cl 10g/L，K₂HPO₄·3H₂O 1g/L，MgSO₄·7H₂O 1g/L，ZnSO₄·7H₂O 1g/L，MnSO₄·H₂O 0.15g/L，CaCl₂ 1g/L

The pH value of the fermentation is 6.5-7.2, and the fermentation liquor is used after being sterilized for 20min at 115 ℃.