阅读说明：本技术 一种定点突变改造的褐藻胶裂解酶突变体及其应用 (Site-directed mutagenesis modified alginate lyase mutant and application thereof ) 是由 史劲松 李恒 李星霖 吴雯 龚劲松 于 2020-03-31 设计创作，主要内容包括：本发明公开了一种定点突变改造的褐藻胶裂解酶突变体及其应用,属于酶工程技术领域。本发明的褐藻胶裂解酶突变体是将氨基酸序列如SEQ ID NO.1所示的褐藻胶裂解酶的第212位谷氨酸替换为组氨酸和/或第222位精氨酸替换为赖氨酸。本发明通过分子对接模拟确定褐藻胶裂解酶活性中心氨基酸并进行定点饱和突变改变蛋白质分子活性位点附近氨基酸残基,提高褐藻胶裂解酶催化效率,进一步提高褐藻胶裂解酶产量。本发明构建的褐藻胶裂解酶分泌能力增强的重组大肠杆菌可将褐藻胶裂解酶酶活较出发菌株提高2.83倍。改造后的基因工程菌产酶能力显著提高,摇瓶发酵生产褐藻胶裂解酶酶活达到15000U/mL,更适合工业应用,可降低生产成本,提高生产效率。(The invention discloses an alginate lyase mutant modified by site-specific mutagenesis and application thereof, belonging to the technical field of enzyme engineering.A 212 th glutamic acid of the alginate lyase with an amino acid sequence shown as SEQ ID NO.1 is replaced by histidine and/or a 222 th arginine is replaced by lysine.)

1. The alginate lyase mutant modified by site-directed mutagenesis is characterized in that the alginate lyase mutant is obtained by replacing glutamic acid at the 212 th site of the alginate lyase with histidine and/or replacing arginine at the 222 th site with lysine, wherein the amino acid sequence of the alginate lyase is shown as SEQ ID NO. 1.

2. The mutant alginate lyase as claimed in claim 1, wherein when the amino acid sequence of the alginate lyase shown as SEQ ID No.1 is replaced by histidine at the 212 th glutamic acid position, the amino acid sequence of the mutant is shown as SEQ ID No. 2.

3. The mutant alginate lyase as claimed in claim 1, wherein when arginine at position 222 of the alginate lyase with an amino acid sequence shown in SEQ ID No.1 is replaced by lysine, the amino acid sequence of the mutant is shown in SEQ ID No. 3.

4. The mutant alginate lyase as claimed in claim 1, wherein when the amino acid sequence of the alginate lyase shown as SEQ ID No.1 is replaced by histidine at the 212 th glutamic acid position and lysine at the 222 th arginine position, the amino acid sequence of the mutant is shown as SEQ ID No. 4.

5. A gene encoding the mutant alginate lyase described in any one of claims 1 to 4.

6. An expression plasmid carrying the gene of claim 5.

7. The expression plasmid of claim 6, wherein the plasmid is pET-28a (+).

8. A recombinant bacterium expressing the mutant according to any one of claims 1 to 4.

9. The recombinant strain of claim 8, wherein the recombinant strain is E.coli Rosetta as a host.

10. The use of the recombinant bacterium of claim 8 in the production of a product comprising alginate lyase.

Technical Field

The invention relates to an alginate lyase mutant modified by site-directed mutagenesis and application thereof, belonging to the technical field of enzyme engineering.

Background

Brown algae is one of the three major algae, and the main components are algin, mannitol and laminarin, wherein, the utilization of the mannitol and the laminarin is relatively mature, so that how to efficiently degrade the algin and realize the industrial application is the focus of the current research, the algin is a linear polysaccharide composed of β -D-mannuronic acid (M) and α -L-guluronic acid (G), and the degradation product of the algin has important physiological activity, such as oxidation resistance, tumor resistance, plant root cell growth promotion and the like, so the algin has received wide attention.

The algin lyase (Aly) can degrade algin through a β -elimination reaction mechanism, and forms unsaturated carbon-carbon double bonds at the non-reducing end of a degradation product, thereby generating a special absorption peak under ultraviolet 235nm and being used for product detection, according to the substrate specificity of Aly, the algin lyase can be divided into polyM, polyG and bifunctional algin lyase, according to the enzyme cutting mode, the algin lyase can be divided into endo-type (endo-type) and exo-type (exo-type) algin lyase, the final product of the alginate degradation by the endo-type Aly is Alginate Oligosaccharide (AOs), the polymerization degree of the product is 2-6, the product of the exo-type Aly is single, the final product is monosaccharide, but the report that disaccharide is the minimum enzyme cutting unit, according to the classification of a CAZY database, the algin lyase belongs to polysaccharide lyase (P L), and is specifically divided into seven algin 73725-5, P733-6, P L-7, P L-14, P L-15, P-8517-8217 and P73717 families.

At present, the method for preparing the alginate oligosaccharide is mainly a chemical method such as acidolysis, the preparation conditions are severe and environmental pollution is easily caused, and in contrast, the method for degrading the alginate polysaccharide by a biological method is mild in conditions, easy to control and environment-friendly, so that the development of the alginate lyase which is efficient, high in stability and easy to prepare is necessary. The current research shows that the genetic engineering is the main means for improving the enzyme activity of the alginate lyase. In addition, the conventional methods for increasing the expression level of recombinant proteins include replacement of strong promoters, signal peptides with high secretion ability, and the like.

Different classification modes are provided according to different properties of the alginate lyase: different alginate lyase enzymes according to the specificity of the brown degradation substrate can be divided into three categories: an enzyme for specifically hydrolyzing a mannuronic acid segment (PolyM), an enzyme for specifically degrading a guluronic acid segment (Poly G) and a bifunctional lyase in which both segments can be cleaved. However, the performance of the existing alginate lyase needs to be further improved to meet the requirement of industrial application.

Disclosure of Invention

In order to solve the problems, the invention obtains the mutant with obviously improved catalytic activity and substrate preference through PCR site-specific saturation mutagenesis, and lays a foundation for further industrial application.

The invention aims to provide an alginate lyase mutant, wherein the alginate lyase mutant is obtained by replacing glutamic acid at position 212 of the alginate lyase with histidine and/or replacing arginine at position 222 of the alginate lyase with lysine, the amino acid sequence of which is shown as SEQ ID NO. 1.

Furthermore, when the 212 th glutamic acid of the alginate lyase with the amino acid sequence shown as SEQ ID NO.1 is replaced by histidine, the amino acid sequence of the mutant is shown as SEQ ID NO. 2.

Furthermore, when the 222 th arginine of the alginate lyase with the amino acid sequence shown as SEQ ID NO.1 is replaced by lysine, the amino acid sequence of the mutant is shown as SEQ ID NO. 3.

Further, when the 212 th glutamic acid of the alginate lyase with the amino acid sequence shown as SEQ ID NO.1 is replaced by histidine and the 222 th arginine is replaced by lysine, the amino acid sequence of the mutant is shown as SEQ ID NO. 4.

It is a second object of the present invention to provide a gene encoding the mutant.

The third purpose of the invention is to provide an expression plasmid carrying the gene.

Furthermore, the plasmid takes pET-28a (+) as a vector.

The fourth purpose of the invention is to provide a recombinant bacterium for expressing the mutant.

Furthermore, the recombinant bacterium takes bacteria, fungi or animal and plant cells as hosts.

The fifth purpose of the invention is to provide the application of the recombinant strain in the production of products containing alginate lyase.

The invention has the beneficial effects that:

the recombinant escherichia coli with enhanced secretion capacity of the alginate lyase constructed by the invention can improve the enzyme activity of the alginate lyase by 2.83 times compared with that of a starting strain, obviously improve the enzyme production capacity of the transformed genetically engineered bacteria, and the enzyme activity of the alginate lyase produced by shake flask fermentation reaches 15000U/m L, so that the recombinant escherichia coli is more suitable for industrial application, can reduce the production cost and improve the production efficiency.

Drawings

FIG. 1 is a nucleic acid gel electrophoresis of mutated alginate lyase; m, marker; 1 to are respectively: alg212A, Alg222A, Alg212H, Alg222K, Alg 212H-222K;

FIG. 2 is a gel electrophoresis diagram of alginate lyase proteins in a mutant strain; m, marker; 1 to 3 are respectively: alg212H, Alg222K, Alg 212H-222K;

FIG. 3 shows the activity of alginate lyase when the site-directed mutation is alanine;

FIG. 4 shows the activity of alginate lyase in the case of 212-point mutation;

FIG. 5 shows the activity of alginate lyase in the case of 222-site mutation;

FIG. 6 shows the activity of alginate lyase before and after combined mutation.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Culture medium:

l B liquid culture medium, peptone 10.0 g/L, yeast powder 5.0 g/L5.0.0 g/L.

TB fermentation medium, peptone 12.0 g/L, yeast powder 24.0 g/L, glycerol 4.0 g/L2 PO417 mmol/L, K2HPO 472 mmol/L.

Measuring the enzyme activity of the alginate lyase:

the enzyme activity measuring condition is that 0.1m L enzyme liquid is added into 0.9m L1 percent sodium alginate solution, the mixture is evenly mixed and reacts for 20min at 45 ℃, then 1m L DNS is added into the system, boiling water bath is carried out for 3min to stop the reaction, the volume is quickly determined to 10m L, 200 mu L is absorbed and added into a 96-hole plate, the light absorption value is measured at 520nm, a contrast group adopts deionized water to replace crude enzyme liquid, 1 enzyme activity unit (U) is defined, under the condition, 1m L enzyme liquid produces 1 mu g of enzyme amount required by reducing sugar per minute, glucose aldehyde acid solutions with different concentrations are prepared, 1m L DNS reagent is added for reaction, the light absorption values of the solutions with different concentrations are measured at 520nm, and the enzyme activity is calculated according to a standard curve.

Specific enzyme activity (U/mg) ═ enzyme activity/protein mass

TABLE 1 primer sequences