阅读说明：本技术 一种褐藻胶裂解酶基因及其应用 (Alginate lyase gene and application thereof ) 是由 王学江 李峰 张志凯 迟艳 于 2019-10-16 设计创作，主要内容包括：本发明公开了一种内切褐藻胶裂解酶的基因序列及其应用。本发明还公开利用基因工程的技术方法,将该新褐藻胶裂解酶的基因克隆到大肠杆菌表达载体上,获得可异源表达该酶的大肠杆菌重组菌株,用该菌株异源表达来制备的褐藻胶裂解酶FsAlgB,具有降解褐藻酸钠制备褐藻酸钠寡糖的功能。本发明提供的褐藻胶裂解酶AlgL可广泛应用于化工、农业、食品、饲料添加、医药及海藻遗传工程等领域。(The invention discloses a gene sequence of incision alginate lyase and application thereof. The invention also discloses a technical method utilizing genetic engineering, the gene of the new alginate lyase is cloned to an escherichia coli expression vector to obtain an escherichia coli recombinant strain capable of heterologously expressing the new alginate lyase, and the alginate lyase FsAlgB prepared by heterologously expressing the strain has the function of degrading sodium alginate to prepare sodium alginate oligosaccharides. The algin lyase AlgL provided by the invention can be widely applied to the fields of chemical industry, agriculture, food, feed additives, medicines, seaweed genetic engineering and the like.)

1. An alginate lyase gene, the nucleotide sequence of which is shown as SEQ ID NO. 1.

2. An alginate lyase, the amino acid sequence of which is shown in SEQ ID NO. 2.

3. The genetically engineered bacterium containing the alginate lyase is characterized in that the strain is introduced with an alginate lyase gene, and the nucleotide sequence of the alginate lyase gene is shown as SEQ ID No. 1.

4. The method for constructing the genetically engineered bacteria producing alginate lyase of claim 3, comprising the following steps:

(1) cloning the alginate lyase gene into a plasmid to obtain a recombinant vector;

(2) and transforming the recombinant vector into host bacteria to obtain the genetically engineered bacteria for producing the alginate lyase.

5. The method for constructing the genetically engineered bacterium producing alginate lyase of claim 4, wherein in the step (1), the plasmid is pET21a (+).

6. The method for constructing genetically engineered bacteria producing alginate lyase of claim 4, wherein in the step (2), the host bacteria is Escherichia coli DH5 α.

7. The use of the alginate lyase of claim 1 for cleaving alginate and fucoidan.

8. The use of the genetically engineered bacterium of claim 3 for producing alginate lyase in fermentation production of alginate lyase.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an alginate lyase gene and application thereof.

Background

China has a wide sea area, and abundant marine biological resources, particularly seaweed resources, are contained in the sea area. The brown algae mainly comprises kelp, undaria pinnatifida and the like, the algin rich in cell walls is a straight-chain acidic polysaccharide, and in a natural state, the algin mainly exists in water-soluble sodium alginate (sodium alginate), potassium alginate and other alginate salts and water-insoluble alginic acid (alginic acid). Sodium alginate (trade name sodium alginate) or other alginates on the market are mainly obtained from brown algae. Researches find that the oligosaccharide obtained by degrading sodium alginate has various biological activities, such as immunoregulation, growth promotion, plant resistance induction, protein stability improvement and the like, so that the oligosaccharide can be widely applied to the fields of chemical industry, agriculture, food, feed addition, medicine and the like (Liuhang, natural product research and development, 2012,24: 201-204). Sodium alginate can be degraded by a variety of methods, including chemical, physical and enzymatic degradation. The chemical degradation method mainly uses acid degradation, but the method has the disadvantages of difficult control of degradation conditions, complex operation and long time consumption. The physical degradation method comprises a radiation method, an ultrasonic method and the like, is generally used together with other degradation methods, has the limit molecular mass of about 50kDa of degradation products, and is difficult to prepare oligosaccharides. The method for degrading sodium alginate by using the alginate lyase has the advantages of mild degradation conditions, high yield and the like, and the alginate lyase gradually becomes a method for preferentially degrading sodium alginate due to the substrate specificity of the enzyme and the capability of providing information for the subsequent research of the chemical structure of oligosaccharide. In addition, the alginate lyase can also be applied to the research in the fields of treatment of cystic fibrosis, seaweed feed processing, seaweed genetic engineering and the like (Wong TY et al, annual Review of Microbiology,2000,54: 289-340).

The alginate lyase is derived from marine animals and plants and various microorganisms (including marine bacteria, soil bacteria and fungi). Sodium alginate lyases can be divided into two broad classes according to their substrate specificity: 1, 4-D-mannuronic acid fragment lyase (EC4.2.2.3) and 1, 4-L-guluronic acid fragment lyase (EC 4.2.2.11). The production of alginate lyase mostly depends on original enzyme-producing animals and plants or microorganisms to obtain enzyme protein, and although a certain amount of enzyme protein can be effectively obtained by the method, the yield is limited, the cost is higher, and the practical application requirements are difficult to meet. With the development of biotechnology, a technical method for efficiently producing alginate lyase by using genetic engineering is provided. The PCR primers of Dong Eun Kim et al were designed based on similar sequences of known related functional genes, and an alginate lyase gene was cloned from Streptomyces sp. ALG-5 strain and successfully expressed in Escherichia coli BL21(DE3) (Kim et al MarineBiotechnology 2009.11: 10-16). By adopting the strategy, the PCR primers can be designed only by knowing the sequence of the related gene to a certain extent, and new molecules in a certain class of proteins with similar structures or functions are found, so that the new gene is difficult to find.

With the continuous development of high-throughput sequencing technology, more and more alginate lyase-producing microbial genome sequences are determined, so that sequence information is analyzed through genome mining technology, the fishing of genes encoding alginate lyase becomes simple and rapid, Badur AH and the like perform sequence determination on the genome of a strain of Vibrio spleendidus derived from the sea, the strain is found to contain 4 genes encoding alginate lyase through analysis, and gene cloning and recombinant expression in Escherichia coli BL21(DE3) are performed (Badur et al. Environ. Microbiol. 2015.81: 1865-pass 1873); most of the currently available alginate lyases are alginate lyases which specifically degrade homopolymannuronic acid, and a few of them are alginate lyases with guluronic acid degrading activity, while the alginate lyases with broad substrate specificity are rare, and only the alginate lyases Aly-SJ02 (Lijianwei, Marine Drugs,2011,21:1374-80) derived from Pseudomonas aeruginosa, the alginate lyases AlyIH (sinusoidal et al, Carbohydratepolymers,2013,98:1476-82) derived from Isoptericola halolerans in termite intestinal tract have low enzymatic activity with broad substrate specificity and poor stability, wherein AlyIH only obtains purified enzyme, and coding genes thereof are not obtained yet, and recombinant expression and molecular modification cannot be carried out; and most of the alginate lyase activity is low. The recombinant alginate lyase has the characteristics of high activity and high stability, and the degradation product is alginate oligosaccharide with low polymerization degree (mainly disaccharide, trisaccharide and tetrasaccharide), so that the recombinant alginate lyase has great industrial application prospect.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a novel alginate lyase.

The technical problem to be solved by the present invention is to provide a genetically engineered bacterium containing the above alginate lyase and a construction method thereof.

The technical problem to be solved by the invention is to provide the application of the alginate lyase.

In order to solve the technical problems, the invention adopts the following technical scheme:

an alginate lyase gene, the nucleotide sequence of which is shown as SEQ ID NO. 1.

An alginate lyase, the amino acid sequence of which is shown in SEQ ID NO. 2.

The genetic engineering bacteria containing the alginate lyase introduce an alginate lyase gene, and the nucleotide sequence of the alginate lyase gene is shown as SEQ ID No. 1.

The construction method of the genetic engineering bacteria for producing the alginate lyase is characterized by comprising the following steps:

(1) cloning the alginate lyase gene into a plasmid to obtain a recombinant vector;

(2) and transforming the recombinant vector into host bacteria to obtain the genetically engineered bacteria for producing the alginate lyase.

In the step (1), the plasmid is pET21a (+).

In the step (2), the host bacterium is Escherichia coli DH5 alpha.

The application of the alginate lyase in the cleavage of alginate and fucoidan is within the protection scope of the invention.

The application of the genetic engineering bacteria of the alginate lyase in the production of the alginate lyase by fermentation is within the protection scope of the invention.

Has the advantages that:

the AlgL is derived from marine bacteria Flammeovirga sp.NJ-04, a DNA sequence for coding the alginate lyase AlgL is obtained by designing a homologous primer, the coding region of the gene is 900bp in length, 299 amino acids are coded, the theoretical molecular weight of the gene is 34.45kDa, and the gene belongs to polysaccharide lyase 7 family. AlgL obtained by recombinant expression of escherichia coli has higher activity on algin, polyM and polyG, and belongs to bifunctional algin lyase. The incision type alginate lyase can be widely applied to the fields of chemical industry, agriculture, food and feed addition, medicine, seaweed genetic engineering and the like.

Drawings

FIG. 1: the result of the alignment of the protein sequence of alginate lyase AlgL.

FIG. 2: the recombinant alginate lyase AlgL expresses a purified polyacrylamide gel electrophoresis image (SDS-PAGE).

FIG. 3 is a graph showing the effect of temperature and pH on the activity and stability of alginate lyase AlgL.

FIG. 4: electrospray ionization mass spectrometry (ESI-MS) analysis chart of products obtained by degrading algin, polyMG, polyM and polyG by using algin lyase.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.