阅读说明：本技术 褐藻胶裂解酶alb02668及基因、重组质粒、工程菌株和在拮抗病原微生物中的应用 (Algin lyase ALB02668, gene, recombinant plasmid, engineering strain and application in antagonistic pathogenic microorganism ) 是由 鲍时翔 黄惠琴 郑志国 朱军 邹潇潇 孟天 胡永华 于 2020-05-06 设计创作，主要内容包括：本发明涉及基因工程技术领域,特别涉及褐藻胶裂解酶ALB02668及基因、重组质粒、工程菌株和在拮抗病原微生物中的应用。该褐藻胶裂解酶的氨基酸序列如SEQ ID NO:1所示。本发明公开了一种新型褐藻胶裂解酶ALB02668及编码该酶的基因序列和氨基酸序列,不但为市场提供了新的褐藻胶裂解酶资源,而且通过公开的基因序列和氨基酸序列,可实现褐藻胶裂解酶的异源表达应用和异源表达生产。本发明开发了利用大肠杆菌重组表达生产褐藻胶裂解酶ALB02668的方法与工艺,利用本方法,不但实现褐藻胶裂解酶ALB02668的重组生产,而且利用Ni-NTA柱特异性吸附表达的蛋白,纯化效率大大提高。(The invention relates to the technical field of genetic engineering, in particular to alginate lyase ALB02668, a gene, a recombinant plasmid, an engineering strain and application in antagonistic pathogenic microorganisms. The amino acid sequence of the alginate lyase is shown as SEQ ID NO. 1. The invention discloses a novel alginate lyase ALB02668 and a gene sequence and an amino acid sequence for coding the novel alginate lyase, which not only provide a novel alginate lyase resource for the market, but also realize the heterologous expression application and the heterologous expression production of the alginate lyase through the disclosed gene sequence and the amino acid sequence. The invention develops a method and a process for producing the algin lyase ALB02668 by utilizing escherichia coli recombinant expression, not only realizes the recombinant production of the algin lyase ALB02668, but also utilizes a Ni-NTA column to specifically adsorb expressed protein, and greatly improves the purification efficiency.)

1. An alginate lyase is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. The gene encoding the alginate lyase of claim 1, wherein the base sequence is represented by SEQ ID NO. 2.

3. An alginate lyase is characterized in that the amino acid sequence thereof is substituted, deleted or added with one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO. 1.

4. A gene encoding the alginate lyase of claim 3.

5. A recombinant plasmid, wherein the recombinant plasmid is a plasmid vector into which the gene of claim 2 or 4 is inserted.

6. The recombinant plasmid of claim 5, wherein the plasmid vector is a pET-28a plasmid vector.

7. An engineered strain, wherein the engineered strain is Escherichia coli into which the recombinant plasmid according to claim 5 or 6 has been transferred.

8. A method for producing the alginate lyase of claim 1 or 3, characterized in that the engineering strain of claim 7 is inoculated in LB culture medium for culture, and centrifugation is carried out after induction expression to obtain supernatant I and thalli;

the thalli is subjected to centrifugation after cracking to obtain supernatant II;

mixing the supernatant I and the supernatant II, purifying the expressed protein by adopting a Ni-NTA column, leaching and eluting to obtain a crude protein solution;

and dialyzing the crude protein solution, and embedding and concentrating by PEG to obtain the alginate lyase.

9. The use of the alginate lyase of claim 1 or 3 for degrading algin or brown algae.

10. The use of the alginate lyase of claim 1 or 3 in degrading exopolysaccharides of pathogenic microorganisms and improving the killing effect of antibiotics on pathogenic microorganisms.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to alginate lyase ALB02668, a gene, a recombinant plasmid, an engineering strain and application in antagonistic pathogenic microorganisms.

Background

Algin is a linear polysaccharide most abundant in brown algae, and comprises two uronic acid monomers of beta-D-mannuronic acid and alpha-L-guluronic acid which are combined in different combination modes through alpha/beta-1, 4 glycosidic bonds to form polyguluronic acid (poly-G), polymannuronic acid (poly-M) and a hybrid segment (poly-GM) formed by random polymerization of guluronic acid and mannuronic acid. The alginate oligosaccharide is an oligomer of algin, has low relative molecular mass, good water solubility, high stability, safety and no toxicity, can be used as a plant growth promoter, an antioxidant, a tumor inhibitor and the like, and has good application prospects in agriculture, food and pharmaceutical industries. At present, the brown algae oligosaccharide is mainly prepared by methods such as an acid method, an oxidative degradation method and an enzymatic method, and the enzymatic preparation has the advantages of mild conditions, easy control, specific product and the like, and becomes one of the current domestic and foreign research hotspots.

Alginate lyase is a kind of polysaccharide lyase, and can generate oligosaccharide products through beta elimination reaction. Alginate lyase can be divided into three types based on substrate specificity, namely polyguluronic acid lyase, polymannuronic acid lyase and polyguluronic acid activity. In terms of mode of action, alginate lyase may be divided into endonuclease and exonuclease, the endonuclease cleaves the internal glycosidic bond of alginate and releases unsaturated oligosaccharides (disaccharide, trisaccharide and tetrasaccharide), and the exonuclease may further degrade oligosaccharides into monomers. The alginate lyase is distributed in 7 polysaccharide lyase families, which are PL-5, PL-6, PL-7, PL-14, PL-15, PL-17 and PL-18 families. The source of the alginate lyase is wide, and marine algae, marine mollusks and microorganisms (including bacteria, fungi and some viruses) and the like have reports on producing the alginate lyase, wherein the reports on the source of the microorganisms are the most. The bacteria mainly include Pseudoalteromonas (Pseudomonas), Vibrio (Vibrio), Flavobacterium (Flavobacterium), Bacillus (Bacillus), Streptomyces (Streptomyces), Paenibacillus (Paenibacillus), Pseudomonas (Pseudomonas), Azotobacter (Azotobacter), Klebsiella (Klebsiella), Corynebacterium (Corynebacterium), Pseudomonas (Alteromonas), Enterobacter (Enterobacter), Sphingomonas (Sphingomonas), and the like. Fungi have been reported less, and Aspergillus crutus, Corouospora intermedia, Dendryphilia arenaria, Dendryphilia salina, and the like have been reported abroad. In 2018, 10 strains of alginate lyase-producing fungi of Rhodotorula (Rhodotorula), Penicillium (Penicillium) and Aspergillus (Aspergillus) were reported to be separated from the sediment of the southern ocean. Suda discovered the gene segment related to alginate lyase in chlorella virus gene for the first time in 1999, and characterized the protein expressed by the gene.

The alginate lyase not only has wide application in the field of alginate oligosaccharide preparation, but also is concerned by people in the field of medicine. Since 1928, penicillin was discovered and antibiotics represented by penicillin saved countless lives from ill hands, and brought great changes to the treatment of infectious diseases. Antibiotics are toxic to bacteria by inhibiting the synthesis of vital substances such as cell walls, cell membranes, nucleic acids, or proteins. However, the long-term use of antibiotics has led to the emergence of antibiotic-resistant strains, such as those resistant to antibiotics like β -lactams, aminoglycosides, chloramphenicol, and tetracycline, and is on the rise. In recent years, the mortality and treatment costs due to bacterial infections caused by staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, and the like have also been increasing. Antibiotic resistance evaluation reports by the uk government 2016 show that about 70 million people die worldwide each year from drug-resistant bacterial infections, and that up to 1000 million deaths may occur by 2050. The world health organization and other regulatory agencies have announced that antibiotic resistance is posing a threat to global health, and new methods for treating bacterial infections are being discovered. The research at home and abroad finds that the biofilm which is synthesized by bacteria and attached to the surface of the biofilm is a natural barrier which is formed by the bacteria in the growth process for adapting to the living environment, and has the protection function. The pathogenic bacteria biomembrane can prevent bacteria from escaping from the immune system of organism and inhibiting phagocytosis of cells, and can prevent penetration of antibiotic to reduce the sensitivity of pathogenic bacteria to antibiotic. The polysaccharide is an important component of the bacterial biofilm, degrades the bacterial biofilm polysaccharide and is a new way for improving the curative effect of antibiotics.

The algin lyase of the invention is derived from Bacillus like HB172198(Paenibacillus sp.) separated and obtained by the applicant, the strain is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is as follows: CGMCC No.15412, the preservation date is: 03 month 05 in 2018. The applicant has already filed a patent for this strain and its use (a B.sp. strain HB172198 and its use, application No. 201810891281.2). In the patent, the applicant applies for protection on the application of a strain HB172198, a fermentation enzyme production process of the strain HB172198 and an enzyme produced by the strain HB172198 in degradation of brown algae. Although the alginate lyase can be obtained by the fermentation method of the bacillus like HB172198, the composition of the fermentation product is complex, the amino acid sequence and the spatial structure of the alginate lyase are not known, the purity of the alginate lyase in the obtained fermentation product is low, and the separation and purification cost is high. Therefore, how to obtain high-purity alginate lyase under the condition of reducing the cost of separation and purification becomes a technical problem which needs to be overcome by researchers.

Disclosure of Invention

In view of the above, the invention provides alginate lyase ALB02668, and gene, recombinant plasmid, engineering strain and application thereof in antagonizing pathogenic microorganism. The invention obtains the gene of the coding algin lyase ALB02668 and the amino acid sequence of the enzyme through the genome sequencing and bioinformatics technology, and develops the method for producing the enzyme by utilizing the recombinant expression of escherichia coli and the application method thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an alginate lyase, and the amino acid sequence of the alginate lyase is shown as SEQ ID NO. 1.

The invention also provides a gene for coding the algin lyase, and the base sequence of the gene is shown as SEQ ID NO. 2.

The invention also provides an alginate lyase, and the amino acid sequence of the alginate lyase is substituted, deleted or added with one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO. 1.

The invention also provides a gene for coding the alginate lyase; the amino acid sequence of the alginate lyase is substituted, deleted and added with one or more amino acids on the basis of the amino acid sequence shown in SEQID NO. 1.

The invention also provides a recombinant plasmid, which is a plasmid vector inserted with the gene.

Preferably, the plasmid vector is a pET-28a plasmid vector.

The invention also provides an engineering strain, wherein the engineering strain is escherichia coli transformed with the obtained recombinant plasmid.

Preferably, Escherichia coli is Escherichia coli BL21(DE 3).

The invention also provides a method for producing the alginate lyase, which comprises the steps of inoculating the obtained engineering strain into an LB culture medium for culture, and centrifuging after induced expression to obtain a supernatant I and thalli;

the thalli is subjected to centrifugation after cracking to obtain supernatant II;

mixing the supernatant I and the supernatant II, purifying the expressed protein by adopting a Ni-NTA column, leaching and eluting to obtain a crude protein solution;

and dialyzing the crude protein solution, and embedding and concentrating by PEG to obtain the alginate lyase.

The invention also provides the application of the alginate lyase in degrading algin or brown algae.

The invention also provides the application of the algin lyase in degrading the exopolysaccharide of pathogenic microorganisms and improving the killing effect of antibiotics on the pathogenic microorganisms.

The invention provides alginate lyase ALB02668, and gene, recombinant plasmid, engineering strain and application in antagonizing pathogenic microorganism. The amino acid sequence of the alginate lyase is shown as SEQ ID NO. 1. The invention has the following beneficial effects:

1. the invention discloses a novel alginate lyase ALB02668 and a gene sequence and an amino acid sequence for coding the novel alginate lyase, which not only provide a novel alginate lyase resource for the market, but also realize the heterologous expression application and the heterologous expression production of the alginate lyase through the disclosed gene sequence and the amino acid sequence.

2. The method and the process for producing the algin lyase ALB02668 by recombinant expression of escherichia coli are developed, the recombinant production of the algin lyase ALB02668 is realized by the method, the expressed protein is specifically adsorbed by a Ni-NTA column, and the purification efficiency is greatly improved.

3. Production of alginate oligosaccharide by using alginate lyase ALB02668

4. The algin lyase ALB02668 is used in combination with antibiotics to improve the killing effect on pathogenic microorganisms, and provides a new technology for killing pathogenic microorganisms.

Drawings

FIG. 1 shows the three-dimensional structure of alginate lyase ALB 02668;

FIG. 2 shows the detection of the induced expression electrophoresis of alginate lyase ALB 02668; drawing notes: protein marker (14-120kD) on the left; the middle is non-induced histone; the right side is induced histone;

FIG. 3 TLC detection of alginate oligosaccharides; note: 1: a pentasaccharide; 2: a trisaccharide; 3: a monosaccharide; 4: and (4) performing enzymolysis on the product.

Detailed Description

The invention discloses algin lyase ALB02668, and a gene, a recombinant plasmid, an engineering strain and application thereof in antagonistic pathogenic microorganism. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The applicant of the invention obtains the gene of the coding algin lyase ALB02668 and the amino acid sequence of the enzyme through the genome sequencing and bioinformatics technology, and develops the method for producing the enzyme by utilizing the recombinant expression of escherichia coli and the application method thereof, and the details are as follows:

1. obtaining an amino acid sequence of the alginate lyase by genome sequencing and bioinformatics technology;

2. producing algin lyase ALB02668 by Escherichia coli recombinant expression;

3. degrading algin and brown algae by using algin lyase ALB02668 to prepare alginate oligosaccharide;

4. the alginate lyase is utilized to degrade extracellular polysaccharide of pathogenic microorganisms, and the killing effect of antibiotics on pathogenic microorganisms is improved.

The algin lyase ALB02668 provided by the invention, and the gene, recombinant plasmid, engineering strain and reagent or instrument used in the application of antagonistic pathogenic microorganism can be purchased from the market.

The invention is further illustrated by the following examples: