1. A strain of Chromobacterium persicinum (Persicobacter sp.) JZB09 was deposited at the China center for type culture Collection, address: eight-channel Wuhan university No. 299 in Wuchang district, Wuhan city, Hubei province, with a preservation date of 2021, 4 months and 12 days and a preservation number of CCTCC M2021354.

2. A method for culturing of the strain Persicobacter sp JZB09 according to claim 1, comprising the steps of:

(1) inoculating the myrothecium roridum JZB09 into a liquid culture medium, and culturing for 16-24 h at the temperature of 25-30 ℃ and the rotating speed of 180-220 r/min to obtain an activated strain;

(2) inoculating the activated strain prepared in the step (1) into a liquid culture medium according to the volume percentage of 1-3%, and culturing for 16-24 h under the conditions that the temperature is 25-30 ℃ and the rotating speed is 180-220 r/min to prepare a seed solution;

(3) inoculating the seed solution prepared in the step (2) into a liquid culture medium according to the volume percentage of 1-5%, and performing amplification culture for 4-6 days under the conditions that the temperature is 25-30 ℃ and the rotating speed is 180-220 r/min to obtain a peach bacillus JZB09 bacterial solution;

preferably, the liquid culture medium in the steps (1), (2) and (3) comprises the following components per liter:

4g/L of tryptone, 2.5g/L of yeast extract, 7.5g/L of potassium chloride, 20g/L of sodium chloride, 1.1g/L of calcium chloride, 7.2g/L of magnesium sulfate, 1.5g/L of ammonium chloride and the balance of water, wherein the pH value is 7.0-7.5.

3. Use of the strain of Chromobacterium persicinum (Persicobacter sp.) JZB09 according to claim 1 for preparing alginate lyase rAly 06925;

preferably, the use of the strain of Chromobacterium persicinum (Persicobacter sp.) JZB09 of claim 1 for degrading algin or alginate oligosaccharides.

4. An alginate lyase rAly06925 derived from peach bacillus (Persicobacter sp.) JZB09, the amino acid sequence of which is shown in SEQ ID NO. 2;

preferably, the alginate lyase rAly06925 coding gene aly06925 has the nucleotide sequence shown in SEQ ID NO. 1;

preferably, the recombinant expression vector I comprises a coding gene aly06925 of the alginate lyase rAly 06925;

preferably, the recombinant bacterium I contains a coding gene aly06925 of the alginate lyase rAly 06925.

5. The use of the coding gene aly06925, recombinant expression vector I, recombinant bacteria I of the alginate lyase rAly06925 of claim 4 in the preparation of the alginate lyase rAly 06925;

preferably, the alginate lyase rAly06925 in claim 4 for degradation of alginate or alginate oligosaccharide;

preferably, the use of the alginate lyase rAly06925 in claim 4 for degrading alginate or alginate oligosaccharides to produce a series of G-rich unsaturated oligosaccharide fragments with delta M at the non-reducing end;

preferably, the use of the alginate lyase rAly06925 in claim 4 for degrading alginate or alginate oligosaccharides to produce unsaturated disaccharides.

6. An alginate lyase rAly06925 mutant enzyme is characterized in that the amino acid mutation site is that the 48 th amino acid of an amino acid sequence SEQ ID NO.2 is changed from tyrosine to alanine;

preferably, the coding gene of the algin lyase rAly06925 mutant enzyme carries out site-directed mutagenesis on the coding gene SEQ ID NO.1 according to the mutation site of the amino acid;

preferably, a recombinant expression vector II comprises a gene encoding the mutant enzyme;

preferably, the recombinant bacterium II comprises a gene coding for the mutant enzyme.

7. The use of the coding gene, recombinant expression vector II and recombinant bacterium II of the mutant enzyme rAly06925 of the alginate lyase in the preparation of the mutant enzyme rAly 06925;

preferably, the claim 6 of the alginate lyase rAly06925 mutant enzyme in degradation of alginate or alginate oligosaccharides;

preferably, the use of the mutant enzyme of alginate lyase rAly06925 in claim 6 in degrading alginate or alginate oligosaccharides to produce a series of G-rich unsaturated oligosaccharide fragments with delta M at the non-reducing end.

8. An algin lyase rAly06925 mutant enzyme, characterized in that the amino acid mutation site is that the 53 th amino acid of the amino acid sequence SEQ ID NO.2 is changed from isoleucine to alanine, the 52 th amino acid is changed from phenylalanine to alanine, the 115 th amino acid is changed from histidine to alanine, the 118 th amino acid is changed from phenylalanine to alanine, the 119 th amino acid is changed from asparagine to alanine or the 243 th amino acid is changed from histidine to alanine;

preferably, the coding gene of the algin lyase rAly06925 mutant enzyme carries out site-directed mutagenesis on the coding gene SEQ ID NO.1 according to the mutation site of the amino acid;

preferably, a recombinant expression vector III, comprising the mutant enzyme encoding gene;

preferably, the recombinant bacterium III contains a gene encoding the mutant enzyme.

9. The use of the coding gene, recombinant expression vector III and recombinant bacterium III of the algin lyase rAly06925 mutant as defined in claim 8 for the preparation of the algin lyase rAly06925 mutant;

preferably, the use of the mutant enzyme of alginate lyase rAly06925 in the degradation of alginate or alginate oligosaccharide;

preferably, the use of the mutant enzyme of alginate lyase rAly06925 in claim 8 in degrading alginate or alginate oligosaccharides to produce a series of G-rich unsaturated oligosaccharide fragments with delta M at the non-reducing end.

10. An alginate lyase rAly06925 mutant enzyme is characterized in that an amino acid mutation site is a 1 st to 65 th amino acid in a truncated amino acid sequence SEQ ID NO. 2;

preferably, the coding gene of the algin lyase rAly06925 mutant enzyme carries out site-directed mutagenesis on the coding gene SEQ ID NO.1 according to the mutation site of the amino acid;

preferably, the recombinant expression vector IV comprises a coding gene of the mutant enzyme;

preferably, the recombinant bacterium IV comprises a coding gene of the mutant enzyme.

11. The use of the coding gene, recombinant expression vector IV and recombinant bacterium IV of the algin lyase rAly06925 mutant as defined in claim 10 for preparing the algin lyase rAly06925 mutant;

preferably, the use of the mutant enzyme of alginate lyase rAly06925 in claim 10 for degrading alginate or alginate oligosaccharide.

12. An alginate lyase rAly06925 mutant enzyme is characterized in that an amino acid mutation site is an amino acid from 45 th position to 60 th position in a truncated amino acid sequence SEQ ID NO. 2;

preferably, the coding gene of the algin lyase rAly06925 mutant enzyme carries out site-directed mutagenesis on the coding gene SEQ ID NO.1 according to the mutation site of the amino acid;

preferably, a recombinant expression vector V, comprising the mutant enzyme encoding gene;

preferably, the recombinant bacterium V contains a gene coding for the mutant enzyme.

13. The use of the coding gene of the algin lyase rAly06925 mutant enzyme, the recombinant expression vector V and the recombinant strain V of claim 12 in the preparation of the algin lyase rAly06925 mutant enzyme;

preferably, the claim of claim 12 on the alginate lyase rAly06925 mutant enzyme in degradation of alginate or alginate oligosaccharides.

14. An algin lyase rAly06925 mutant enzyme is characterized in that the amino acid mutation site is that the 244 th amino acid in the amino acid sequence SEQ ID NO.2 is mutated into other amino acids except aromatic hydrocarbon amino acid;

preferably, the coding gene of the algin lyase rAly06925 mutant enzyme carries out site-directed mutagenesis on the coding gene SEQ ID NO.1 according to the mutation site of the amino acid;

preferably, a recombinant expression vector VI comprises a coding gene of the mutant enzyme;

preferably, the recombinant bacterium VI contains a coding gene of the mutant enzyme.

15. The use of the coding gene, recombinant expression vector VI and recombinant bacterium VI of the mutant enzyme rAly06925 of the alginate lyase in claim 14 for preparing the mutant enzyme rAly 06925;

preferably, the use of the mutant enzyme of alginate lyase rAly06925 in claim 14 for degrading alginate or alginate oligosaccharide.

Background

The algin is a linear anionic polysaccharide formed by randomly linking two sugar units of beta-D-Mannuronate (M) and alpha-L-guluronic acid (alpha-L-Guluronate, G) through beta-1, 4 glycosidic bonds, and M and G are epimers with each other at C5 position^[1]. The algin is mainly produced from cell wall and cytoplasm mesenchyme of large brown algae such as herba Zosterae Marinae, Sargassum, Fucus vesiculosus and Macrocystis^[2]. In addition, the conditional pathogens Pseudomonas aeruginosa and certain soil microorganisms such as azotobacter vinelandii can also secrete algin^[3,4]The difference is that the algin of microbial origin has acetylation modification^[5]. The algin is nontoxic and harmless, and can be used for removing heavy metal ions in vivo in food industry^[6]And increasing the consistency of ice cream^[7]It can also be used in hemostatic gauze, hemostatic agent and bandage in pharmaceutical industry^[8]And drug-encapsulating material^[9]. The algin, agar and carrageenan are the three kinds of ocean polysaccharide with the largest yield, the highest economic value and the most extensive application. For a long time, the high-valued research of algin has been one of the key points in the field of marine biotechnology. The research shows that the algin oligose changes the nuclear morphology of leukemia cell U973 to cause apoptosis^[10]And the inhibition effect on leukemia cells U973 is realized. In addition, recent studies have shown that "mannooligosaccharide diacid (GV 971)" prepared from poly-M alginate oligosaccharides inhibits beta-amyloid precipitation, cytotoxicity and aggregation of cells^[11]Can be used for treating mild and moderate AlzheimerAlzheimer Disease (AD). Therefore, the algin oligosaccharide with specific M/G ratio and polymerization degree has important application value and economic value, and has important significance for realizing the high-efficiency preparation of the oligosaccharide.

Alginate Lyase is a class of Polysaccharide Lyases (PL) that catalyze the cleavage of glycosidic bonds by β -elimination reaction, and form C4 ═ C5 unsaturated double bonds at the non-reducing end of the oligosaccharide product, and a conjugated structure with C5-position C ═ O (carboxygroup), thereby producing an oligosaccharide product containing an unsaturated end (Δ) and having a characteristic absorption near 232nm^[12]. The source of the alginate lyase is very wide, and comprises soil microorganisms, marine algae, marine mollusks, echinoderms and various marine microorganisms living on the body surface or in vivo, wherein bacteria are the main source of the alginate lyase^[13,14]. However, limited by the low yield of natural alginate lyase and the influence of the combination factors of poor water solubility and activity of most of the enzymes despite the high yield of transgenic alginate lyase, studies on substrate selectivity, substrate degradation pattern and oligosaccharide production characteristics of the enzymes have been few so far, and only G-specific endonuclease Aly5 is shown^[15]M-specific endonucleases Pae-AlgL and Avi-AlgL^[16]G-biased endonuclease Aly1^[17]And Aly2^[18]And the like. Thus, overall, insufficient information on the enzymatic properties is provided, leading to a lack of tool enzymes and to inaccurate use. Especially in industry, the incision type alginate lyase is a tool enzyme for efficiently preparing series alginate oligosaccharide products.

The inventor's prior research focuses on the analysis of the incision type alginate lyase, and has focused on the substrate selectivity, the substrate degradation mode and the oligosaccharide production characteristics of a series of incision type alginate lyase, and summarized by induction, the substrate selectivity and the substrate degradation mode of the enzyme are found to determine the oligosaccharide production characteristics of the incision enzyme. It has been found that the alginate endonuclease has the structure characteristic of the oligosaccharide end product with the succession rule of delta M to delta G or delta G to delta M^[15-18]G fragment-rich monosaccharide exo-alginate lyase specially producing delta G tail end^[19]And producing G-rich fragments of the.DELTA.M-terminusM-specific broad-spectrum polysaccharide degrading enzyme with algin as optimal substrate in PL8 family^[20]However, no report on the algin lyase derived from Persicobacter strain, characteristics and application value is found at present, and especially the algin endonuclease specially producing a delta M terminal end product is not found.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides peach color bacillus JZB09, alginate lyase rAly06925, and a coding gene and application of the enzyme.

In the following contents, delta is unsaturated monosaccharide, and X is G or M; m is beta-1, 4-D-mannuronic acid, and G is alpha-1, 4-L-guluronic acid.

The algin lyase rAly06925 is abbreviated as "rAly 06925".

The enzyme can specifically degrade mannuronic acid oligosaccharide fragments from algin, and is M-specific algin lyase. Therefore, M-rich fragments, namely M-MMXn, G-MMXn and delta-MMXn (n is more than or equal to 1 and is a natural number; X is G or M) are specifically degraded when the algin is degraded, and the position of a glycosidic bond shown in the-M is efficiently cut, so that a series of unsaturated oligosaccharide final products with non-reducing ends only delta M are generated. Therefore, rAly06925 is the first reported endonuclease-type alginate lyase specially produced only with delta M terminals. Therefore, the method can be used for completely degrading the alginate polysaccharide or the alginate oligosaccharide to specifically prepare the series of unsaturated oligosaccharide fragments which take Delta M as the non-reducing end and are rich in G. In addition, the influence of the enzyme and the serial mutants thereof on the enzyme activity is deeply discussed, and the potential catalytic motif N is determined²³⁸-N²³⁹-H²⁴⁰-G²⁴¹-T²⁴²-H²⁴³And Y is⁸⁷，Q¹⁷⁰，H²⁴⁰，Y²⁹⁵Etc. are key catalytic site residues. In particular, the extra peptide fragment K of rAly06925 is truncated⁴⁵-N⁶⁰(i.e., T45-60N, which is the amino acid from position 45 to position 60 in the truncated amino acid sequence) or Y²⁴⁴The mutation of other amino acids except aromatic hydrocarbon amino acid results in the loss of water solubility of the recombinant protein, so that the additional peptide segment K⁴⁵-N⁶⁰And contains Y²⁴⁴Residues have a key role in maintaining protein conformation or determining the hydrophilicity of the protein surface.

The invention is realized by the following technical scheme:

a strain of Chromobacterium persicinum (Persicobacter sp.) JZB09 was deposited at the China center for type culture Collection, address: the preservation date of the Wuhan university No. 299 in the eight routes of the Wuhan district of the Wuhan city in Hubei province is 2021, 4 months and 12 days, and the preservation number is CCTCC M2021354.

"Chromobacterium persicae (Persicobacter sp.) JZB 09" is simply referred to as "Chromobacterium persicae JZB 09".

The method for culturing the myrothecium roridum JZB09 comprises the following steps:

(1) inoculating the myrothecium roridum JZB09 into a liquid culture medium, and culturing for 16-24 h at the temperature of 25-30 ℃ and the rotating speed of 180-220 r/min to obtain an activated strain;

(2) inoculating the activated strain prepared in the step (1) into a liquid culture medium according to the volume percentage of 1-3%, and culturing for 16-24 h under the conditions that the temperature is 25-30 ℃ and the rotating speed is 180-220 r/min to prepare a seed solution;

(3) inoculating the seed solution prepared in the step (2) into a liquid culture medium according to the volume percentage of 1-5%, and performing amplification culture for 4-6 days under the conditions that the temperature is 25-30 ℃ and the rotating speed is 180-220 r/min to obtain the peach bacillus JZB09 bacterial solution.

Preferably, the liquid culture medium in the steps (1), (2) and (3) comprises the following components per liter:

4g/L of tryptone, 2.5g/L of yeast extract, 7.5g/L of potassium chloride, 20g/L of sodium chloride, 1.1g/L of calcium chloride, 7.2g/L of magnesium sulfate, 1.5g/L of ammonium chloride and the balance of water, wherein the pH value is 7.0-7.5.

The application of the peach color bacillus JZB09 in preparing the alginate lyase rAly 06925.

Use of the above-mentioned strain of Chromobacterium persicum (Persicobacter sp.) JZB09 for degrading algin or alginate oligosaccharides.

An alginate lyase rAly06925 derived from peach color bacillus JZB09, the amino acid sequence of which is shown in SEQ ID NO. 2.

The Alginate lyase rAly06925 only contains one structural domain, namely Alginate _ lyase.

The nucleotide sequence of the coding gene aly06925 of the alginate lyase rAly06925 is shown in SEQ ID NO. 1.

The total length of the coding gene aly06925 is 1197bp, and the coded protein contains 399 amino acids and has a molecular weight of about 45.6 kDa.

A recombinant expression vector I contains the coding gene aly06925 of the algin lyase rAly 06925.

A recombinant bacterium I comprises the coding gene aly06925 of the alginate lyase rAly 06925.

The coding gene aly06925, the recombinant expression vector I and the recombinant bacterium I of the algin lyase rAly06925 are applied to the preparation of the algin lyase rAly 06925.

The application of the algin lyase rAly06925 in degrading algin or algin oligosaccharide.

According to the invention, the preferred application of the alginate lyase rAly06925 is in degrading algin or alginate oligosaccharides to produce a series of G-rich unsaturated oligosaccharide fragments with delta M at the non-reducing end.

According to the invention, the preferable application of the alginate lyase rAly06925 is in degrading the alginate or the alginate oligosaccharide to produce unsaturated disaccharide.

An alginate lyase rAly06925 mutant enzyme, wherein the amino acid mutation site is that the 48 th amino acid of an amino acid sequence SEQ ID NO.2 is changed from tyrosine to alanine;

preferably, the coding gene of the algin lyase rAly06925 mutant enzyme carries out site-directed mutagenesis on the coding gene SEQ ID NO.1 according to the mutation site of the amino acid;

preferably, a recombinant expression vector II comprises a gene encoding the mutant enzyme;

preferably, the recombinant bacterium II comprises a gene coding for the mutant enzyme.

The coding gene of the algin lyase rAly06925 mutant enzyme, a recombinant expression vector II and a recombinant bacterium II are applied to the preparation of the algin lyase rAly06925 mutant enzyme.

The application of the mutant enzyme rAly06925 in degrading algin or algin oligosaccharide.

According to the invention, the preferred application of the above-mentioned alginate lyase rAly06925 mutant enzyme in degrading alginate or alginate oligosaccharide to produce a series of G-rich unsaturated oligosaccharide fragments with delta M at the non-reducing end is provided.

An algin lyase rAly06925 mutant enzyme, wherein the amino acid mutation site is that the 53 th amino acid of the amino acid sequence SEQ ID NO.2 is changed from isoleucine to alanine, the 52 th amino acid is changed from phenylalanine to alanine, the 115 th amino acid is changed from histidine to alanine, the 118 th amino acid is changed from phenylalanine to alanine, the 119 th amino acid is changed from asparagine to alanine or the 243 th amino acid is changed from histidine to alanine.

Preferably, the coding gene of the algin lyase rAly06925 mutant enzyme carries out site-directed mutagenesis on the coding gene SEQ ID NO.1 according to the mutation site of the amino acid;

preferably, a recombinant expression vector III, comprising the mutant enzyme encoding gene;

preferably, the recombinant bacterium III contains a gene encoding the mutant enzyme.

The coding gene, recombinant expression vector III and recombinant bacterium III of the algin lyase rAly06925 mutant enzyme are applied to preparation of the algin lyase rAly06925 mutant enzyme

The application of the mutant enzyme rAly06925 in degrading algin or algin oligosaccharide.

According to the invention, the preferred application of the above-mentioned alginate lyase rAly06925 mutant enzyme in degrading alginate or alginate oligosaccharide to produce a series of G-rich unsaturated oligosaccharide fragments with delta M at the non-reducing end is provided.

An alginate lyase rAly06925 mutant enzyme, wherein the amino acid mutation site is the amino acid from 1 st to 65 th in a truncated amino acid sequence SEQ ID NO. 2;

preferably, the coding gene of the algin lyase rAly06925 mutant enzyme carries out site-directed mutagenesis on the coding gene SEQ ID NO.1 according to the mutation site of the amino acid;

preferably, the recombinant expression vector IV comprises a coding gene of the mutant enzyme;

preferably, the recombinant bacterium IV comprises a coding gene of the mutant enzyme.

The coding gene, the recombinant expression vector IV and the recombinant bacterium IV of the algin lyase rAly06925 mutant enzyme are applied to the preparation of the algin lyase rAly06925 mutant enzyme.

The application of the mutant enzyme rAly06925 in degrading algin or algin oligosaccharide.

An alginate lyase rAly06925 mutant enzyme, wherein the amino acid mutation site is the amino acid from 45 th position to 60 th position in a truncated amino acid sequence SEQ ID NO. 2;

preferably, the coding gene of the algin lyase rAly06925 mutant enzyme carries out site-directed mutagenesis on the coding gene SEQ ID NO.1 according to the mutation site of the amino acid;

preferably, a recombinant expression vector V, comprising the mutant enzyme encoding gene;

preferably, the recombinant bacterium V contains a gene coding for the mutant enzyme.

The coding gene of the algin lyase rAly06925 mutant enzyme, a recombinant expression vector V and a recombinant bacterium V are applied to the preparation of the algin lyase rAly06925 mutant enzyme.

The application of the mutant enzyme rAly06925 in degrading algin or algin oligosaccharide.

An algin lyase rAly06925 mutant enzyme, wherein the amino acid mutation site is that the 244 th amino acid in the amino acid sequence SEQ ID NO.2 is mutated into other amino acids except aromatic hydrocarbon amino acid;

preferably, the coding gene of the algin lyase rAly06925 mutant enzyme carries out site-directed mutagenesis on the coding gene SEQ ID NO.1 according to the mutation site of the amino acid;

preferably, a recombinant expression vector VI comprises a coding gene of the mutant enzyme;

preferably, the recombinant bacterium VI contains a coding gene of the mutant enzyme.

The coding gene of the algin lyase rAly06925 mutant enzyme, a recombinant expression vector VI and a recombinant bacterium VI are applied to preparation of the algin lyase rAly06925 mutant enzyme.

The application of the mutant enzyme rAly06925 in degrading algin or algin oligosaccharide.

The amino acid sequence SEQ ID NO.2 has Met as amino acids from position 1 to position 65¹～Ala⁶⁵。

The amino acids from 45 th position to 60 th position in the amino acid sequence SEQ ID NO.2 are K⁴⁵-N⁶⁰I.e. Lys⁴⁵～Asn⁶⁰。

The non-catalytic domain T65N of the algin lyase rAly06925 (T65N is Met in the amino acid sequence)¹～Ala⁶⁵Non-catalytic domain portion) plays an essential role in maintaining protein conformation or determining hydrophilicity of protein surface and exerting catalytic function.

The catalytic motif of the alginate lyase rAly06925 is N²³⁸-N²³⁹-H²⁴⁰-G²⁴¹-T²⁴²-H²⁴³And Y is⁸⁷，Q¹⁷⁰，H²⁴⁰，Y²⁹⁵Etc. are key catalytic site residues. In particular, the extra peptide fragment K of rAly06925 is truncated⁴⁵-N⁶⁰Or a reaction of Y²⁴⁴The mutation of other amino acids except aromatic hydrocarbon amino acid results in the loss of water solubility of the recombinant protein, so that the additional peptide segment K⁴⁵-N⁶⁰And contains Y²⁴⁴Residues have a key role in maintaining protein conformation or determining the hydrophilicity of the protein surface.

Advantageous effects

1. The invention discloses an alginate lyase rAly06925 obtained from the genome of peach bacillus (Persicobacter sp.) JZB09 for the first time, reports the characteristics and application value of the alginate lyase from peach bacillus for the first time, and is an endo-type alginate lyase specially produced only containing delta M tail end for the first time.

2. The activity of the alginate lyase rAly06925 degraded alginase prepared by the invention is 135U/mg, and the method is suitable for producing series unsaturated oligosaccharides.

3. When the alginate lyase rAly06925 prepared by the invention degrades alginate polysaccharide formed by randomly mixing M, G, only M-MMXn, G-MMXn, delta-MMXn (n is more than or equal to 1 and is a natural number; X, M or G) and other M-rich motifs can be specifically identified, and the position of a glycoside bond shown in the M-rich motif can be efficiently cut, so that a series of unsaturated oligosaccharide final products with a non-reducing end only being delta M are generated. Therefore, the method can be used for degrading the algin or algin oligosaccharide to produce a series of G-rich unsaturated oligosaccharide fragments and unsaturated monosaccharide delta, wherein the non-reducing end of the series contains delta M.

4. The serial mutants of the alginate lyase rAly06925 prepared by the invention disclose that the catalytic motif of the enzyme is N²³⁸-N²³⁹-H²⁴⁰-G²⁴¹-T²⁴²-H²⁴³And Y is⁸⁷，Q¹⁷⁰，H²⁴⁰，Y²⁹⁵Etc. are key catalytic site residues. In particular, the extra peptide fragment K of rAly06925 is truncated⁴⁵-N⁶⁰Or a reaction of Y²⁴⁴The mutation of other amino acids except aromatic hydrocarbon amino acid results in the loss of water solubility of the recombinant protein, so that the additional peptide segment K⁴⁵-N⁶⁰And contains Y²⁴⁴Residues have a key role in maintaining protein conformation or determining the hydrophilicity of the protein surface. The conserved motif N of the alginate lyase rAly06925 is preliminarily determined²³⁸-N²³⁹-H²⁴⁰-G²⁴¹-T²⁴²-H²⁴³And key active site residue Y⁸⁷，Q¹⁷⁰，H²⁴⁰，Y²⁹⁵，Y²⁴⁴It is found that the catalytic mechanism is novel and needs to be explained in detail by structural biology research, which is different from the identified alginate lyase family. The novel alginate lyase provides effective supplement for the cognition of the novel alginate lyase family and the conserved motif, and provides certain theoretical revelation for related enzymological research.

Drawings

FIG. 1 is a BLASTp analysis result diagram (A) composed of functional modules of alginate lyase rAly06925 and an alginate lyase system evolution analysis diagram (B) with higher consistency of partial identified functional characterization and unidentified functional characterization;

in the figure: aly06925 is alginate lyase rAly 06925;

aly06925-T65N is a truncated body rAly 06925-T65N.

FIG. 2 is a diagram showing the multi-sequence alignment analysis of alginate lyase rAly06925 and alginate lyase of unknown function;

in the figure: aly06925 is alginate lyase rAly 06925.

FIG. 3 is a polyacrylamide gel electrophoresis (SDS-PAGE) chart showing the expression and purification of alginate lyase rAly06925 and truncation rAly 06925-T65N;

in the figure: a is alginate lyase rAly06925

M and protein molecular weight standard, wherein the sizes of the bands from top to bottom are respectively: 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4 kD; lane 1, control strain wall-broken whole strain liquid, loading 4. mu.L; lane 2, the whole bacterial liquid after the wall breaking of the recombinant bacteria, the loading amount of which is 4 mu L; lane 3 recombinant bacteria wall-broken supernatant, 4. mu.L of sample; lane 4, rAly06925 purified by nickel column, loading 2. mu.L;

b is a truncated body rAly06925-T65N

M and protein molecular weight standard, wherein the sizes of the bands from top to bottom are respectively: 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4 kD; lane 1, control strain wall-broken whole strain liquid, loading 4. mu.L; lane 2, the whole bacterial liquid after the wall breaking of the recombinant bacteria, the loading amount of which is 4 mu L; lane 3 recombinant bacteria wall-broken supernatant, 4. mu.L of sample; lane 4, truncated rAly06925-T65N purified by nickel column, 2. mu.L loading.

FIG. 4 is an HPLC analysis chart of the enzyme activity detection of alginate lyase rAly06925 and truncation rAly 06925-T65N;

in the figure: UDP2, an unsaturated disaccharide; UDP3, unsaturated trisaccharide; UDP4, unsaturated tetrasaccharide; UDP5, unsaturated pentasaccharide; UDP6, unsaturated hexasaccharide; e (-) is a negative control with no enzyme added.

FIG. 5 is a graph showing the effect of temperature on the activity of alginate lyase rAly 06925.

FIG. 6 is a graph showing the effect of temperature on the stability of alginate lyase rAly 06925.

FIG. 7 is a graph showing the effect of pH on the activity of alginate lyase rAly 06925.

FIG. 8 is a graph showing the effect of pH on stability of alginate lyase rAly 06925.

FIG. 9 is a bar graph showing the effect of metal ions and chemicals on the activity of alginate lyase rAly 06925.

FIG. 10 is a graph showing the effect of NaCl concentration on the activity of alginate lyase rAly 06925.

FIG. 11, molecular gel chromatography-HPLC analysis of oligosaccharide product during degradation of alginate by alginate lyase rAly 06925.

FIG. 12 is a HPLC analysis chart (A) and (B) of the unsaturated oligosaccharide fragments UDP2, UDP3, UDP4, UDP5 and UDP6 prepared after completely degrading algin with the algin lyase rAly06925¹H-NMR chart (B).

FIG. 13 is a HPLC analysis chart of alginate lyase rAly06925 completely degrading a series of saturated oligosaccharides M3-M7(A) and G5 (B).

FIG. 14, HPLC analysis chart of alginate lyase rAly06925 completely degrading saturated oligosaccharide M3-M6 whose reducing end is labeled with 2-AB;

in the figure: (-) is a negative control with no enzyme added.

FIG. 15, HPLC check chart of degradation of the characteristic unsaturated oligosaccharide UDP3-UDP6 with alginate lyase rAly 06925.

FIG. 16 is a drawing showing a three-dimensional mimic structure (A) and a comparison drawing (B) of alginate lyase rAly 06925;

in fig. B: red is rAly06925, green is Pae-AlgL of the structure identified in PL5, and yellow is an extra peptide stretch out of rAly06925 (T45-60N).

FIG. 17 is a bar graph of the relative enzyme activity analysis of alginate lyase rAly06925 truncation T45-60N and its mutants.

FIG. 18, HPLC (A) of alginate lyase rAly06925 mutant I53A degradation end product and¹H-NMR (B) analysis chart.

Detailed Description

The following examples are set forth so as to provide a thorough disclosure of some of the commonly used techniques of how the present invention may be practiced, and are not intended to limit the scope of the invention. The inventors have made the best effort to ensure accuracy with respect to parameters (e.g., amounts, temperature, etc.) used in the examples, but some experimental errors and deviations should be accounted for. Unless otherwise indicated, molecular weight in the present invention refers to weight average molecular weight and temperature is in degrees Celsius.

Source of biological material

Chromobacterium persicae (Persicobacter sp.) JZB09 was deposited in the center of chinese type culture collection, address: eight Wuhan university No. 299 in Wuhan district, Wuhan city, Hubei province, with preservation date of 2021 year 4 and 12 days and preservation number of CCTCC M2021354.

Algin used in the present invention was purchased from Sigma, imidazole from Amresco, anthranilamide from Sigma-aldrich, and algin series saturated oligosaccharide from Qingdao Bozhi Virginian Biotech Co.

Example 1

Extraction of genomic DNA of strain JZB09, Persicobacter persicae (Persicobacter sp.)

The inventor collects sea mud samples from the red island accessory tidal flat of gulf of Qingdao, glue and China in 10 months of 2009, and screens the samples by using a unique carbon source method to obtain a strain of polysaccharide degrading bacteria JZB09, which is identified as peach bacillus (Persicobacter sp.) JZB09 through molecules. Inoculating strain of Chromobacterium persicinum (Persicobacter sp.) JZB09 into M10 liquid medium, culturing at 28 deg.C and 200rpm under shaking to 600nm absorbance (OD)₆₀₀) Is 1.18; 20mL of the culture broth was centrifuged at 28 ℃ and 12,000 Xg (g, gravity constant) for 20min, and the bacterial pellet was collected.

The M10 liquid culture medium comprises the following components: 4g/L of tryptone, 2.5g/L of yeast extract, 7.5g/L of potassium chloride, 20g/L of sodium chloride, 1.1g/L of calcium chloride, 7.2g/L of magnesium sulfate, 1.5g/L of ammonium chloride and the balance of water, and the pH value is 7.2.

Adding 12.0mL of lysozyme buffer solution into the thallus sediment to obtain about 14.0mL of bacterial liquid, and respectively adding 560 mu L of lysozyme with the concentration of 20mg/mL to obtain the final concentration of about 800 mu g/mL; placing in ice water bath for 1.0h, transferring to 37 deg.C water bath, and warm-bathing for 2h until the reaction system is viscous; adding 0.82m L hexadecyl sodium sulfonate solution with the concentration of 100mg/mL and 60 mu L proteinase K solution with the concentration of 100mg/m L, and bathing for 1.0h at the temperature of 52 ℃; adding 15mL of Tris-balanced phenol/chloroform/isoprene (volume ratio is 25: 24: 1), and slightly inverting and mixing until full emulsification; centrifuging at 4 deg.C for 10min at 10,000 Xg, collecting supernatant, adding 2.0M L NaAc-HAc (pH 5.2,3.0M) buffer solution and 17.0mL absolute ethanol (stored at-20 deg.C), and mixing; picking out filamentous DNA with a gun head, transferring to a centrifugal tube of 1.5m L, washing for 2 times with 70% ethanol, and discarding supernatant after microcentrifugation; centrifuging at 10,000 Xg and 4 deg.C for 2min, and completely discarding supernatant; the DNA precipitate was air-dried in a sterile bench, and then the DNA sample was dissolved with sterile deionized water overnight at 4 ℃ to prepare genomic DNA.

Example 2

Scanning of genome of strain JZB09 of Chromobacterium persicae (Persicobacter sp.) and sequence analysis thereof

Scanning sequencing of the genomic DNA prepared in example 1 was carried out by pyrosequencing technology, and was carried out by Meiji Biochemical, Shanghai. The DNA sequencing results were analyzed with the online software of the NCBI (National Center for Biotechnology information, ttp:// www.ncbi.nlm.nih.gov /) website. The analytical software used for the NCBI website is Open Reading Frame Finder (ORF Finder, http:// www.ncbi.nlm.nih.gov/gorf. html.) and Basic Local Alignment Search Tool (BLAST, http:// BLAST. NCBI. nlm. nih. gov/BLAST. cgi).

The result of analysis by the above biological software shows that the genomic DNA of strain No. JZB09 of Achromobacter persicae (Persicobacter sp.) carries 1 candidate alginate lyase gene aly06925, the coded alginate lyase rAly06925 has a full length of 1197bp, the nucleotide sequence of the coded alginate lyase gene is shown as SEQ ID NO.1, the coded alginate lyase gene rAly contains 399 amino acids, the amino acids at the 1-18 th positions of the N-end are I type signal peptides, and the amino acid sequence of the coded alginate lyase gene is shown as SEQ ID NO. 2. On-line analysis using BLAST software revealed that the Alginate Lyase rAly06925 contained a putative domain, Alginate _ Lyase superfamily (as shown in FIG. 1-A), which was identical to the identified Alginate Lyase (Azotoba)The sequence identity of AlgL of PL-5 family derived from cter vinelandii) was the greatest, 9%. Phylogenetic tree analysis shows that rAly06925 has a close relationship with PL-5 and PL-17 families (as shown in FIG. 1-B), but does not cluster with identified PL-5 or PL-17 family members, but clusters with alginate lyase with unidentified functional characteristics, and is located in a branch, so that the rAly06925 is classified into a polysaccharide lyase new family which is not reported. Based on the multiple sequence comparison with the alginate lyase with higher sequence identity which is not identified to be functionally characterized at present, rAly06925 contains two potential catalytic motifs (shown in FIG. 2), namely N²³⁸-N²³⁹-H²⁴⁰-G²⁴¹-T²⁴²-H²⁴³、N¹¹³-N¹¹⁴-H¹¹⁵-T¹¹⁶-D¹¹⁷-F¹¹⁸。

Example 3

Recombinant expression of gene aly06925 and truncation aly06925-T65N in E.coli BL21(DE3) strain

PCR amplification was performed using the genomic DNA prepared in example 1 as a template. The primer sequences are as follows:

the forward primer rAly 06925-F: 5' -gCATATGCAAAAAACCATTTCATTAACGG-3’(Nde I)，SEQ ID NO.3；

Reverse primer rAly 06925-R: 5' -gCTCGAGTTGGAAAAACGCTTCCACGAAATTCC-3’(Xho I)，SEQ ID NO.4；

The forward primer rAly 06925-T65N-F: 5'-GTTACGCATAAAACGGGTGTTCCACC-3', SEQ ID NO. 5;

reverse primer rAly 06925-T65N-R: 5'-CATATGTATATCTCCTTCTTAAAGTTAAAC-3', SEQ ID NO. 6.

The restriction enzyme Nde I site is underlined in the forward primer rAly06925-F, and the restriction enzyme Xho I site is underlined in the reverse primer rAly 06925-R. The high fidelity DNA polymerase PrimeSTAR HS DNA Polymer used was purchased from Dalibao, China, and the PCR reagents used were operated according to the product instructions provided by this company.

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 30 seconds; annealing at 65 ℃ for 30 s; extension at 72 ℃ for 170 s; 35 cycles; extending for 10min at 72 ℃; stabilizing at 5 deg.c for 10 min.

Connecting the PCR product with pEASY-Blunt simple vector, transforming into Escherichia coli Trans1-T1 strain, coating on Luria-Bertani culture medium solid plate containing 50 ug/mL kanamycin, culturing at 37 ℃ for 16h, picking out single clone; inoculating the single clone into a liquid Luria-Bertani culture medium containing 50 mu g/mL kanamycin for culture, and extracting plasmids; carrying out PCR verification on the plasmid by using an amplification primer; carrying out double enzyme digestion on the recombinant plasmid which is verified to be correct by Nde I and Xho I, and connecting the recombinant plasmid with a pET30a plasmid vector which is also subjected to double enzyme digestion under the catalysis of DNA ligase; the ligation product is transformed into an Escherichia coli DH5 alpha strain, the strain is spread on a Luria-Bertani culture medium solid plate containing 100 mu g/mL kanamycin, and after culturing for 16h at 37 ℃, a single clone is picked; inoculating the single clone into a liquid Luria-Bertani culture medium containing 100 mu g/mL kanamycin for culture, and extracting plasmids; carrying out double enzyme digestion verification on the plasmid, obtaining an amplification product with a correct size direction as a result, and preliminarily proving that the constructed recombinant plasmid is correct; the recombinant plasmid was then sequenced, and it was confirmed that the gene aly06925 represented by SEQ ID No.1 was inserted between the Nde I and Xho I cleavage sites of pET30a in the correct direction, and thus the construction of the recombinant plasmid was correct, and the recombinant plasmid was named pET30a-Aly 06925.

In order to verify the function of the non-catalytic domain of the alginate lyase rAly06925, the functional module of the alginate lyase rAly06925 shown in figure 1 is subjected to T65N (the Met in the amino acid sequence is cut off)¹～Ala⁶⁵Non-catalytic domain portion). In order to obtain a recombinant plasmid of a truncated body rAly06925-T65N, taking a recombinant plasmid pET30a-Aly06925 as a template, carrying out PCR amplification by using primers rAly06925-T65N-F and rAly06925-T65N-R, and carrying out terminal phosphorylation and cyclization connection after recovering PCR product gel; transforming the ligation product to Escherichia coli DH5 alpha, coating the ligation product on an LB culture medium solid plate containing 50 mu g/mL Kana, culturing at 37 ℃ for 14h, and picking a monoclonal; inoculating the single clone into a liquid LB culture medium containing 50 mu g/mL Kana for culture, and performing shake culture at 37 ℃ for 12 h; PCR verification is carried out by using an amplification primer, the constructed recombinant plasmid is proved to be correct primarily, and a small plasmid extraction reagent is usedExtracting plasmid from the cassette (Tiangen Biochemical technology Co., Ltd.); the recombinant plasmid was then sequenced to verify that the correct size and orientation of the desired fragment was inserted into the recombinant plasmid, and the successfully constructed recombinant plasmid was designated pET30a-Aly 06925-T65N. But is an inclusion body after induction expression, then double enzyme digestion is carried out on recombinant plasmid pET30a-Aly06925-T65N by using restriction enzymes Nde I and Xho I, and the fragment of the enzyme digestion product is recovered by agarose gel electrophoresis; the pCold TF plasmid is digested by restriction enzymes Nde I and Xho I, the fragments of the digested products are recovered by agarose gel electrophoresis, and then the two fragments are connected under the catalysis of DNA ligase; transforming the ligation product to Escherichia coli DH5 alpha, spreading on LB culture medium solid plate containing 50 ug/mLAmp, performing inverted culture at 37 deg.C for 14h, and picking out single clone; inoculating the single clone into a liquid LB culture medium containing 50 mu g/mLAmp, and performing shake culture at 37 ℃ for 12 h; PCR verification is carried out by using an amplification primer, the constructed recombinant plasmid is preliminarily proved to be correct, and plasmid extraction is carried out by using a small plasmid extraction kit (Tiangen Biochemical technology Co., Ltd.); the recombinant plasmid was then sequenced to verify the size and orientation of the inserted gene sequence in the recombinant plasmid, and the successfully constructed recombinant plasmid was named pColdTF-Aly 06925-T65N.

Recombinant plasmids pET30a-Aly06925, pET30a and pCold TF-Aly06925-T65N were transformed into E.coli strain BL21(DE3) (purchased from Invitrogen, USA), and then the inducible expression of alginate lyase rAly06925 and truncation rAly06925-T65N was performed using isopropyl thiogalactoside (IPTG) according to the procedures provided by the same. Centrifuging at 8,000 Xg and 4 deg.C for 15min, collecting thallus, resuspending thallus with buffer solution A, and ultrasonicating in ice water bath. Further centrifugation was carried out at 15,000 Xg at 4 ℃ for 30min to collect water-soluble fractions, and algin lyase rAly06925 and rAly06925-T65N were purified with Ni-Sepharose. Gradient elution was performed with buffer A (50mM Tris, 150mM NaCl, pH 8.0) containing 10, 50, 250, 500mM imidazole, and the purification conditions were as per the gel's product manual. And detecting the purification condition of the recombinase by polyacrylamide gel electrophoresis. The results are shown in FIG. 3-A: after the recombinant plasmid pET30a-Aly06925 is subjected to IPTG induced expression in an E.coli BL21(DE3) strain, the water-soluble product accounts for 95 percent, the alginate lyase rAly06925 purified by nickel column affinity chromatography is single strip on electrophoresis, and the position of the single strip is matched with the predicted molecular weight; and (3) putting the purified samples of the alginate lyase rAly06925 and rAly06925-T65N into a dialysis bag with the minimum molecular interception amount of 10kDa, dialyzing at the environment of 4 ℃ to obtain the alginate lyase rAly06925 enzyme solution with the enzyme solution concentration of 3 mu g/mu L and the truncation rAly06925-T65N enzyme solution with the enzyme solution concentration of 2 mu g/mu L, and carrying out subsequent experiments by using the enzyme solution.

Example 4

Activity verification of alginate lyase rAly06925 and truncation rAly06925-T65N

The alginate substrate solution prepared by deionized water with the mass volume concentration of 12g/L, the alginate lyase rAly06925 enzyme solution or the truncated rAly06925-T65N enzyme solution prepared in example 3, and 150mM HAc-NaAc (pH 6.0) buffer solution are mixed in a proportion of 1: 1: 1 (volume ratio) and reacting for 4 hours at 30 ℃. Heating the reaction product in boiling water bath for 10min to inactivate enzyme, transferring to ice water bath for 5min, centrifuging at 12,000 Xg and 4 deg.C for 15min, collecting supernatant, and performing HPLC analysis. With NH at a concentration of 0.20M₄HCO₃The solution was equilibrated with Superdex Peptide 10/300GL (GE) molecular gel chromatography column at a flow rate of 0.40mL/min for at least 2 beds. A part of the reaction supernatant obtained above was loaded with 20. mu.g/sample by an auto-sampler under the same conditions as the other conditions, and detected at 232 nm.

The results are shown in FIG. 4, alginate lyase rAly06925 can degrade algin well, but the truncated form of alginate lyase rAly06925-T65N cannot degrade algin, which indicates that the non-catalytic domain of rAly06925 plays an essential role in maintaining protein conformation or determining the hydrophilicity and the catalytic function of the protein surface. Furthermore, the enzyme activity of rAly06925 was about 135U/mg as determined by the DNS reducing sugar method.

Example 5

Determination of optimum temperature of alginate lyase rAly06925

Preparing alginate substrate with mass volume concentration of 12g/L with deionized water, sterilizing, cooling to room temperature, and standing at 0 deg.CIncubating in a water bath environment at 10 deg.C, 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C for 30 min. To 100. mu.L of the substrate solution were added 30. mu.L of the alginate lyase rAly06925 prepared in example 3, 100. mu.L of 150mM HAc-NaAc (pH 6.0) buffer solution and 70. mu.L of deionized water, and the mixture was mixed and reacted for 4 hours. 3 parallel samples at each temperature were used as controls with a boiling water bath inactivated recombinase preparation. The concentration (OD) of newly formed reducing sugar in each reaction system was measured by the DNS-reducing sugar method₅₄₀) And calculating the average value, and performing deviation analysis. The reaction temperature corresponding to the maximum absorbance is the optimal temperature of the recombinase, and the relative enzyme activity (RA) is defined as: percentage of each absorption value to the maximum absorption value.

The results are shown in FIG. 5: when the enzyme activity is measured by taking the algin as a substrate, the algin lyase rAly06925 achieves the maximum activity when reacting at 35 ℃, which shows that the optimum reaction temperature of the algin lyase rAly06925 is 35 ℃. Compared with other temperatures, the alginate lyase rAly06925 is an alginate lyase suitable for marine environment, and the relative enzyme activity is more than 80% only at 30-35 ℃.

Example 6

Temperature stability analysis of alginate lyase rAly06925

Treating the alginate lyase rAly06925 prepared in example 3 at different temperatures (0-60 ℃) for 0.5h, 1h, 2h, 4h, 12h and 24h respectively, and then mixing the treated product with an alginate substrate prepared from distilled water and having a mass volume concentration of 12g/L according to a volume ratio of 1: 9, then reacting for 4 hours at the optimal temperature and measuring the residual enzyme activity, setting 3 parallel samples at each temperature, and taking the alginate lyase preparation inactivated by boiling water bath as a control group. The enzyme activity of the enzyme solution without heat treatment is defined as 100% relative activity.

The results are shown in FIG. 6: after pretreatment for 12h at the temperature lower than 20 ℃ or pretreatment for 2h at the temperature lower than 30 ℃, the alginate lyase rAly06925 still has more than 50 percent of residual enzyme activity; after pretreatment is carried out for 2 hours at the temperature of more than 40 ℃, the residual enzyme activity of the alginate lyase rAly06925 is sharply reduced, and the higher the temperature is, the faster the enzyme activity is reduced, which shows that the alginate lyase rAly06925 has certain thermal stability.

Example 7

Determination of optimum pH and stability of alginate lyase rAly06925

Respectively using NaAc-HAc buffer solution and NaH with the concentration of 50mM₂PO₄-Na₂HPO₄Buffer solution and Tris-HCl buffer solution are respectively mixed with algin to prepare an algin substrate with the mass volume concentration of 12g/L, the corresponding pH values are respectively 5, 6, 7, 8, 9 and 10, and each pH value is adjusted at the optimum temperature. Dissolving the substrate, placing the substrate in the optimum temperature for incubation for 30min, adding 30 mu L of the alginate lyase rAly06925 prepared in example 3, 100 mu L of the above buffer solution and 70 mu L of deionized water into each 100 mu L of the substrate solution, mixing uniformly, and continuing to react for 4 h. 3 parallel samples under each pH condition, and the alginate lyase preparation inactivated in boiling water bath was used as a control group. The concentration of newly formed reducing sugar (OD) in each reaction system was measured by the DNS-reducing sugar method₅₄₀) And calculating the average value, and performing deviation analysis. The reaction pH corresponding to the maximum absorption value is the optimum pH of the alginate lyase, and the relative enzyme activity (RA) is defined as: percentage of each absorption value to the maximum absorption value.

Meanwhile, 30 mu L of the alginate lyase rAly06925 prepared in the embodiment 3 and 100 mu L of the buffer solutions are mixed uniformly, the mixture is placed in an environment at 35 ℃ for respective incubation for 2h, then the mixture is mixed with 100 mu L of the alginate substrate with the mass volume concentration of 12g/L, the mixture is placed at the optimal temperature for respective reaction for 4h, and the residual enzyme activity is measured. 3 parallel samples under each pH condition, and the alginate lyase preparation inactivated in boiling water bath was used as a control group. The concentration of newly formed reducing sugar (OD) in each reaction system was measured by the DNS-reducing sugar method₅₄₀) And calculating the average value, and performing deviation analysis. The enzyme activity of the enzyme solution without pretreatment is defined as 100% relative activity.

As shown in FIG. 7, the optimum reaction pH of the alginate lyase rAly06925 was 6.0, and the activity of rAly06925 was drastically reduced in a pH meta-acid or a basic environment, which indicates that the enzyme was suitable for a buffer condition of a neutral meta-acid and was not suitable for a severe environment such as a strong acid or a strong base. After pretreatment for 2h by different buffers, the activity of the alginate lyase rAly06925 is still kept above 50% within the pH range of 6.0-8.0; the residual enzyme activity of the alginate lyase rAly06925 at pH5.0, pH9.0 and pH10.0 is reduced rapidly to less than 20%, which indicates that the alginate lyase rAly06925 has certain pH tolerance and is more suitable for neutral environment, as shown in FIG. 8.

Example 8

Effect of Metal ions and chemical reagents on the Activity of alginate lyase rAly06925

Preparing an alginate substrate with the mass volume concentration of 12g/L by using deionized water, the alginate lyase rAly06925 prepared in example 3 and water according to the volume ratio of 5: 1: 4 to a final concentration of 1mM or 10mM, and then reacted at 35 ℃ for 4 hours while setting 3 parallel groups per sample. The enzyme activity was determined according to the DNS-reducing sugar method described previously. The control group was rAly06925 activity without any metal ions or chemicals, set at 100%.

The results are shown in FIG. 9, 1mM Ca²⁺1mM DTT (dithiothreitol) has obvious promotion effect on the enzyme activity of the alginate lyase rAly06925, and the relative enzyme activity is improved by about 25%; k⁺、Li⁺、Na⁺、Mg²⁺And a chemical reagent Glyerol has no obvious influence on the enzyme activity of the alginate lyase rAly 06925; and Ag at a concentration of 1mM or 10mM⁺、Cu²⁺、Hg²⁺、Cr³⁺、Mn²⁺、Ni²⁺、Zn²⁺、Fe²⁺、Fe³⁺The isoheavy metal ions and chemical reagents SDS, EDTA and imidazole have obvious inhibition effect on the enzyme activity of the alginate lyase rAly 06925; furthermore 1mM Co²⁺、10mM Pb²⁺Also has obvious inhibition effect on the enzyme activity of the algin lyase rAly 06925.

Example 9

Effect of NaCl on Activity of alginate lyase rAly06925

Preparing 5M NaCl mother liquor by deionized water, adding 30 mul of alginate lyase rAly06925 prepared in example 3 and 100 mul of 150mM HAc-NaAc (pH 6.0) buffer solution into each 100 mul of substrate solution, adding 70 mul of NaCl mother liquor diluted by proper amount of deionized water to make the final concentration of NaCl in the reaction system be 0M, 0.2M, 0.4M, 0.6M, 0.8M and 1M respectively, placing at the optimum temperature for each reaction for 4h, setting 3 parallel groups at each concentration, and using the enzyme preparation inactivated by boiling water bath as a control group. The enzyme activity was determined according to the DNS-reducing sugar method described previously. The control group was the activity of rAly06925 in the absence of NaCl, set at 100%.

The results are shown in FIG. 10, when the NaCl concentration is 0-0.2M, the alginate lyase rAly06925 activity increased with the increase of NaCl concentration; but when the concentration of NaCl is 0.2-1.0M, the enzyme activity of the alginate lyase rAly06925 is reduced along with the increase of the concentration of NaCl; wherein 0.2M NaCl has the highest promotion effect on the alginate lyase rAly06925, so that the relative enzyme activity of the alginate lyase rAly06925 is improved by about 60 percent, and the enzyme activity of the alginate lyase rAly06925 is kept above 50 percent within the range of 0-0.6M; this indicates that alginate lyase rAly06925 has certain NaCl tolerance.

Example 10

High Performance Liquid Chromatography (HPLC) analysis of product of degrading algin by algin lyase rAly06925

100 mul of each of algin with the mass volume concentration of 12g/L and NaAc-HAc (pH 6.0) buffer solution with the mass volume concentration of 150mM are prepared by deionized water, 70 mul of deionized water is added to the mixture to be mixed evenly, and the mixture is incubated for 1h at 35 ℃. And 30. mu.L of the alginate lyase rAly06925 prepared in example 3 was added, and the mixture was mixed well and then reacted further, with sampling at intervals. Heating the reaction product in boiling water bath for 10min, and transferring into ice water bath for 5 min. The mixture was centrifuged at 12,000 Xg at 4 ℃ for 15min, and the supernatant was collected.

With NH at a concentration of 0.20M₄HCO₃The solution was equilibrated with Superdex Peptide 10/300GL (GE) molecular gel chromatography column at a flow rate of 0.40mL/min for at least 2 beds. The sample subjected to the algin enzymolysis is loaded with 20 mu g/sample by an automatic sample injector, and the other conditions are unchanged, and the sample is detected at 232 nm. The integrated area of each oligosaccharide component was analyzed using HPLC operating software to calculate the relative molarity. As shown in FIG. 11, under the above conditions, alginate lyase rAly06925 produced oligosaccharides with larger molecular weight initially when degrading the alginate substrate, and the oligosaccharide product with 235nm characteristic absorption peak with prolonged reaction timeThe content gradually increases and eventually tends to stabilize. The main products of the final reaction were five oligosaccharide products with peak times of 41.8 ', 39.1 ', 36.5 ', 34.7 ' and 33.4 ' in HPLC assay, and after reference to the peak times of the molecular weight standards, the oligosaccharide products were determined to correspond to UDP2, UDP3, UDP4, UDP5 and UDP6, respectively, in a molar ratio of about 6:4:3:2: 1. The results show that the alginate lyase rAly06925 is an incision type alginate lyase.

Example 11

Degradation of final product of algin by algin lyase rAly06925¹H-NMR spectroscopy

According to the most suitable reaction system, degrading 36mg of algin completely by using algin lyase rAly06925, and after the reaction is finished, carrying out a series of treatments on the sample to obtain supernatant filtrate. Using molecular gel chromatography column Superdex^TMPeptide 10/300GL (GE company) was used to separate and purify the final product of alginate lyase rAly06925, which was completely degraded by alginate, and the purity was checked by HPLC after the combination, and the chromatographic conditions were as in example 10. As shown in FIG. 12-A, the final product purity was above 99%. Then freeze-drying for desalination, replacing deuterium with hydrogen by heavy water, and carrying out¹H-NMR detection analyzes the structural characteristics of the oligosaccharide product. As a result, as shown in FIG. 12-B, the unsaturated oligosaccharide products of the series all had a characteristic absorption peak at 5.57ppm, and only a.DELTA.M signal peak was observed, and no.DELTA.G signal peak was observed. This indicates that the non-reducing end of the final oligosaccharide product obtained by degrading M/G poly-segment algin by rAly06925 is in delta M structure.

Example 12

Degradation characteristic of alginate lyase rAly06925 for degrading alginate saturated oligosaccharide substrate

To further reveal the oligosaccharide-producing properties of rAly06925, the enzyme was systematically analyzed for substrate selectivity and oligosaccharide substrate degradation pattern. Taking solutions of equimolar amounts of saturated oligosaccharides including poly-M-segment oligosaccharides (M3-M7) and poly-G-segment oligosaccharides (G3-G7), reacting the solutions with alginate lyase rAly06925 under the optimal condition for 24h, carrying out HPLC detection after treating the reaction solution, determining the polymerization degree of each oligosaccharide product by referring to a molecular weight standard, analyzing the integral area of each oligosaccharide component, calculating the relative molar concentration, and referring to example 10 under the chromatographic condition. The results are shown in fig. 13-a, which is significant in degrading M-series saturated oligosaccharides, but not M3. Degradation of M7, M6, M5 produced mainly UM2 and UM3 and a portion of UM4, and degradation of M4 produced mainly UM3 and a portion of UM 2. The G series saturated oligosaccharides were not degraded (only the results of the alginate lyase rAly06925 was shown here after degradation of G5), as shown in FIG. 13-B.

The results show that alginate lyase rAly06925 is M-specific alginate endonuclease, M4 is the minimal saturated oligosaccharide substrate of alginate lyase rAly06925, and M is the minimal saturated oligosaccharide product of rAly 06925.

Example 13

Degradation characteristic of alginate lyase rAly06925 for degrading fluorescently-labeled alginate saturated oligosaccharide substrate

About 10. mu.g of saturated poly-M oligosaccharide was added to a solution of dimethyl sulfoxide (DMSO) containing excess anthranilamide (2-AB) and sodium borohydride, mixed well and incubated in a 60 ℃ water bath for 2 h. Spin-dry to dryness, add 500. mu.L of deionized water to dissolve the sample, shake the sample with 200. mu.L of chloroform, centrifuge, and collect the supernatant. Repeatedly extracting with chloroform for at least 7 times to obtain reaction solution with reducing end labeled by fluorescence. Saturated poly-M trisaccharide (2AB-M3), tetrasaccharide (2AB-M4), pentasaccharide (2AB-M5) and hexasaccharide (2AB-M6) with reducing ends fluorescently labeled are used as substrates. The substrate and alginate lyase rAly06925 are reacted for 24h under the optimal condition. The reactants were treated and subjected to HPLC detection, the relative molecular weight of each oligosaccharide product was determined with reference to molecular weight standards, the integrated area of each oligosaccharide component was analyzed, and the relative molar concentrations were calculated. HPLC detection conditions: a chromatographic column: superdex^TMPeptide 10/300 GL; a detector: a fluorescence detector with excitation wavelength Ex 330nm and detection wavelength Em 420 nm; mobile phase: 0.2M NH₄HCO₃。

As shown in FIG. 14, alginate lyase rAly06925 degraded 2AB-M6, with the major products being 2AB-UM3 and 2AB-UM4 at a molar ratio of about 1.4:1, and also produced small amounts of 2AB-UM5 and 2AB-UM 2. The main products are 2AB-UM3 and 2AB-UM4 at a molar ratio of about 2:1 when 2AB-M5 is degraded, and a small amount of 2AB-UM2 is produced, and in addition, when 2AB-M4 is not completely degraded, the main product is 2AB-UM3 but 2AB-M3 is not degraded.

The above results show that 2AB-M4 is the minimal synthetic M series saturated oligosaccharide substrate of rAly06925, and M is the minimal saturated oligosaccharide product with a variable substrate degradation pattern. The 2-AB label at the reducing end did not significantly affect the activity of the enzyme, and indicates that the enzyme was degraded from the non-reducing end of the alginate substrate.

Example 14

Degradation characteristic of alginate lyase rAly06925 for degrading alginate unsaturated oligosaccharide substrate

To further investigate the enzymatic properties of this enzyme, we used M-biased alginate exomonosaccharide Aly6 based on the experimental basis available in the subject group^[19]Incompletely degrading algin, and separating with molecular gel chromatographic column Superdex^TMPeptide 10/300GL (GE company) separated and purified the degradation product, prepared a series of characteristic oligosaccharide products with only AG as the non-reducing end. The oligosaccharide fragment and alginate lyase rAly06925 were reacted for 24h under the optimum conditions, the reaction solution was treated and then subjected to HPLC detection, and the chromatographic conditions were as in example 10.

As shown in FIG. 15, the alginate lyase rAly06925 partially degraded UDP6 to produce UDP4, UDP3 and UDP2, slightly degraded UDP5 to produce UDP4 and unsaturated monosaccharide (. DELTA.), and did not degrade UDP4 and UDP 3. The result shows that the alginate lyase rAly06925 degrades the unsaturated oligosaccharide in an endo-mode, the minimum unsaturated oligosaccharide substrate is UDP5, but the degradation is very weak, presumably because the series of unsaturated oligosaccharides are obtained by Aly6 incompletely degrading the alginate, and the non-reducing ends of the unsaturated oligosaccharides are all delta G units through identification, which is consistent with the result that the alginate lyase rAly06925 is an M-specific alginate lyase.

Further, referring to fig. 11, 12, 13 and 15, it is assumed that the oligosaccharide production characteristics when alginate lyase rAly06925 degrades alginate are determined by both substrate selectivity and substrate degradation pattern: when the rAly06925 degrades the algin polysaccharide substrate, M-MMXn, G-MMXn, delta-MMXXn (n is more than or equal to 1 and is a natural number; X, G or M) and the like which are rich in M are specifically degraded, and the position of a glycosidic bond shown in the M is efficiently cut, so that a series of unsaturated oligosaccharide final products with non-reducing ends only delta M are generated.

Example 15

Alginate lyase rAly06925 homologous modeling

Through bioinformatics analysis, the sequence of the alginate lyase rAly06925 is novel, and the amino acid sequence of the alginate lyase (AlgL of PL5 family derived from Azotobacter vinelandii) has the maximum consistency of only 9%. In addition, alginate lyase rAly06925 is evolutionarily special, although it has a recent relationship with PL5 and PL17 families, it does not cluster with the identified PL5 or PL17 family members, but rather stands alone. Through multiple sequence alignment analysis, we found that there are two groups of candidate motifs NNH (N) in the alginate lyase rAly06925 sequence¹¹³-N¹¹⁴-H¹¹⁵And N²³⁸-N²³⁹-H²⁴⁰) And the sequence is not identical with the reported conserved sequence (NNHSYW) of PL5, convincing key catalytic site residues in the sequence cannot be identified through bioinformatics analysis, family classification cannot be directly carried out, and similar research reports are not found. Furthermore, we speculate that rAly06925 may contain other key amino acid residues than NNH. Therefore, the three-dimensional structure MODEL was simulated on line using SWISS-MODEL, and three-dimensional simulation was performed using the Pae-AlgL and AI-III of the identified structures in PL5 as templates, as shown in FIG. 16-A, rAly06925 consists of many α -helical structures and has an α/α sleeve structure, similar to the alginate lyase AlgL identified in PL5 family from Pseudomonas aeruginosa and Azotobacter vinelandii, and contains a tunnel-like catalytic cavity, which is presumed to be the basis for the development of alginate lyase activity. Comparison by PyMOL software found: rAly06925 has one more alpha-helix (K)⁴⁵-N⁶⁰) And some putative key amino acid residues (Y)⁴⁸、F⁵²、I⁵³、Y⁸⁷、H¹¹⁵、F¹¹⁸、N¹¹⁹、Q¹⁷⁰、H²⁴⁰、H²⁴³、Y²⁴⁴、Y²⁹⁵) As shown in fig. 16-B.

Example 16

Molecular modification and catalytic mechanism research of alginate lyase rAly06925

In order to obtain recombinant plasmids of alginate lyase rAly06925 truncation T45-60N, PCR amplification is carried out by taking recombinant plasmid pET30a-Aly06925 as a template and Aly06925-T45-60N-F and Aly06925-T45-60N-R as primers, and after PCR product gel is recovered, 5' terminal phosphorylation is carried out and T4 DNA ligase is used for connection; transforming the ligation product to Escherichia coli DH5 alpha, spreading on LB culture medium solid plate containing 50 ug/mL Kana, culturing at 37 deg.C for 14h, and selecting single clone; inoculating the single clone into a liquid LB culture medium containing 50 mu g/mL Kana for culture, and performing shake culture at 37 ℃ for 12 h; PCR verification is carried out by using an amplification primer, the constructed recombinant plasmid is preliminarily proved to be correct, and plasmid extraction is carried out by using a small plasmid extraction kit (Tiangen Biochemical technology Co., Ltd.); the recombinant plasmid was then sequenced to verify that the correct size and orientation of the desired fragment was inserted into the recombinant plasmid, and the successfully constructed recombinant plasmid was designated pET30a-Aly 06925-T45-60N. After induction expression, the recombinant plasmid pET30a-Aly06925-T45-60N is subjected to double enzyme digestion by using restriction enzymes Nde I and Xho I in the same way, and enzyme digestion product fragments are recovered by agarose gel electrophoresis; the pColdTF plasmid is digested by restriction enzymes Nde I and Xho I, the fragments of the digested product are recovered by agarose gel electrophoresis, and then the two fragments are connected under the catalysis of DNA ligase; transforming the ligation product to Escherichia coli DH5 alpha, spreading on LB culture medium solid plate containing 50 ug/mLAmp, performing inverted culture at 37 deg.C for 14h, and picking out single clone; inoculating the single clone into a liquid LB culture medium containing 50 mu g/mLAmp, and performing shake culture at 37 ℃ for 12 h; PCR verification is carried out by using an amplification primer, the constructed recombinant plasmid is preliminarily proved to be correct, and plasmid extraction is carried out by using a small plasmid extraction kit (Tiangen Biochemical technology Co., Ltd.); the recombinant plasmid was then sequenced to verify the size and orientation of the inserted gene sequence in the recombinant plasmid, and the successfully constructed recombinant plasmid was named pColdTF-Aly 06925-T45-60N.

In order to obtain recombinant plasmids of alginate lyase rAly06925 series mutants, recombinant plasmids pET30a-Aly06925 are used as templates, corresponding mutation primers (table 1) are used, and high-Fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase is used for PCR amplification to obtain amplified fragments; using the enzyme DpnIRemoving the methylated template plasmid in the amplification product; carrying out homologous recombination on the 5 'end and the 3' end of the amplification product under the catalysis of an enzyme Exnase II to complete the cyclization of the amplification product; after the reaction is finished, converting the cyclized amplification product into escherichia coli DH5 alpha, coating the escherichia coli DH5 alpha on an LB culture medium solid plate containing 50 mu g/mL Kana, culturing for 14h at 37 ℃, and selecting a monoclonal; inoculating the single clone into a liquid LB culture medium containing 50 mu g/mL Kana for culture, and performing shake culture at 37 ℃ for 12 h; PCR verification is carried out by using a mutation primer, the constructed recombinant plasmid is preliminarily proved to be correct, and plasmid extraction is carried out by using a small plasmid extraction kit (Tiangen Biochemical technology Co., Ltd.); the recombinant plasmid is then sequenced to verify that the target fragment with the correct size and direction is inserted into the recombinant plasmid, and the successfully constructed recombinant plasmid is named as pET30a-Aly06925-Y48A, pET30a-Aly06925-F52A, pET30a-Aly06925-I53A, pET30a-Aly06925-Y87A, pET30a-Aly06925-H115A, pET30a-Aly06925-F118A, pET30a-Aly06925-N119A, pET30a-Aly06925-Q170A, pET30a-Aly06925-H240A, pET30a-a 063672-H a, pET 30-a-3606925-Y244, pET 30-a-36295. And to Y²⁴⁴Saturation mutation is carried out on the site, and the primer sequences are shown in table 1. The conditions for the induction expression and purification of the series of mutant proteins are as in example 3, and the conditions for the activity detection are as in example 4.

The activity of alginate lyase rAly06925 truncated T45-60N and a series of mutants is detected by using a DNS-reducing sugar method, as shown in figure 17: y in algin lyase rAly06925⁸⁷，Q¹⁷⁰，H²⁴⁰，Y²⁹⁵All inactivated after mutation, suggesting that the residues are key site residues, and the N is confirmed²³⁸-N²³⁹-H²⁴⁰Are conserved motifs. In addition, Y is found²⁴⁴The site mutation is that other amino acids except aromatic amino acid are expressed in inclusion body, and Y is presumed to be²⁴⁴The density of electron clouds of the aromatic groups on the sites is high, and the electron clouds play a key role in maintaining the conformation of the protein or determining the hydrophilicity and hydrophobicity of the surface of the protein. Likewise, extra alpha-helix (K)⁴⁵-N⁶⁰) Plays a key role in maintaining protein conformation and playing a catalytic role. In conclusion, the conserved motif N of the novel alginate lyase rAly06925 is preliminarily determined²³⁸-N²³⁹-H²⁴⁰-G²⁴¹-T²⁴²-H²⁴³And key active site residue Y⁸⁷，Q¹⁷⁰，H²⁴⁰，Y²⁹⁵，Y²⁴⁴It is found that the catalytic mechanism is novel and needs to be explained in detail by structural biology research, which is different from the identified alginate lyase family. The novel alginate lyase provides effective supplement for the cognition of the novel alginate lyase family and the conserved motif, and provides certain theoretical revelation for related enzymological research.

Using molecular gel chromatography column Superdex^TMSeparating and purifying final products obtained by degrading algin by recombinant enzyme rAly06925 mutant I53A by Peptide 10/300GL (GE company), and detecting purity by HPLC after merging; as shown in fig. 18-a, the purity was above 99%; freeze drying for desalting, replacing deuterium with hydrogen in heavy water, and final¹H-NMR detection and analysis by related nuclear magnetic software show that: the series of unsaturated oligosaccharide products all had a characteristic absorption peak at 5.57ppm, with only a Δ M signal peak and no Δ G signal peak being observed, as shown in FIG. 18-B. This indicates that the non-reducing end of the final oligosaccharide product obtained by degrading M/G poly-segment algin with alginate lyase rAly06925 mutant I53A is delta M unit, and the structural characteristics of the final product are not affected by the change of the amino acid residue at the site.

Example 17

Cellulose, microcrystalline cellulose, carboxymethyl cellulose, starch, pectin, agar, mannan, algin, carrageenan, kappa-carrageenan, lambda-carrageenan, tau-carrageenan, Hyaluronic Acid (HA), Chondroitin Sulfate A (CSA), Chondroitin Sulfate C (CSC), Chondroitin Sulfate E (CSE), dermatan sulfate (HS/DS), chitin, Xanthan gum (Xanthan) and the alginate lyase rAly06925 enzyme solution prepared in example 3, 150mM HAc-NaAc (pH 6.0) buffer solution which are prepared by deionized water and have the mass volume concentration of 12g/L respectively are mixed according to the volume ratio of 1: 1: 1, and reacting for 72 hours at 50 ℃. Heating the reaction product in boiling water bath for 10min to inactivate enzyme, transferring into ice water bath for 5min, centrifuging at 12,000 Xg and 4 deg.C for 15min, and collecting supernatant. DNS analysis is performed separately. Mixing a certain volume of supernatant with DNS (3, 5-p-nitroxylene) reaction solution with the same volume, heating in boiling water bath for 10min, cooling to room temperature, and measuring light absorption value at 540 nm; the detection result shows that the alginate lyase rAly06925 has an obvious degradation effect on the alginate and has no degradation effect on other polysaccharides.

The invention discloses an alginate lyase rAly06925 obtained from the genome of Microbacterium persicum (Persicobacter sp.) JZB09 for the first time, wherein the coding gene of the alginate lyase is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2; the method reports the algin lyase from the genus Tacrobium for the first time, and the characteristics and application value of the algin lyase, and reports that the algin lyase specially produces the incision type algin lyase only containing delta M tail end for the first time, and the algin lyase has the advantages of obvious difference with the existing known algin lyase, stable physicochemical property, high activity and potential of industrial application; the enzyme activity of the alginate lyase rAly06925 for degrading the alginate is 135U/mg, and the method is suitable for producing series unsaturated oligosaccharides.

Algin lyase rAly06925 mutant: amino acid 48 from tyrosine to alanine, Y48A; amino acid 53 changed from isoleucine to alanine, i.e., I53A; amino acid position 52 changed from phenylalanine to alanine, i.e., F52A; amino acid 115 changed from histidine to alanine, i.e., H115A; amino acid 118 changed from phenylalanine to alanine, i.e., F118A; the 119 th amino acid is changed from asparagine to alanine, i.e., N119A; the 243 rd amino acid is changed from histidine to alanine, namely H243A; the mutant enzymes were all effective in degrading algin, as shown in FIG. 17.

Algin lyase rAly06925 mutant: the amino acid mutation site is the amino acid from the 1 st to the 65 th position in the truncated amino acid sequence SEQ ID NO.2, namely rAly 06925-T65N; the amino acid mutation site is the amino acid from 45 th position to 60 th position in the truncated amino acid sequence SEQ ID NO.2, namely Aly 06925-T45-60N; or the amino acid mutation site is that the 244 th amino acid in the amino acid sequence SEQ ID NO.2 is mutated into other amino acids except aromatic hydrocarbon amino acid; the mutant is in the form of inclusion body after induced expression, which is beneficial to the extraction and purification of mutant enzyme, and the enzyme in the inclusion body has the enzyme activity of degrading algin.

TABLE 1

Reference to the literature

[1]Chang P S,Mukerjea R,Fulton D B,et al.Action of Azotobacter vinelandii poly-β-d-mannuronic acid C-5-epimerase on synthetic d-glucuronans[J].Carbohydrate research,2000,329(4):913-922.

[2]Kloareg B,Quatrano R S.Structure of the cell walls of marine algae and ecophysiological functions of the matrix polysaccharides[J].OCEANOGRAPHY AND MARINE BIOLOGY:AN ANNUALREVIEW,1988,26:259-315.

[3]Hay I D,Rehman Z U,Moradali M F,et al.Microbial alginate production,modification and its applications[J].Microbial biotechnology,2013,6(6):637-650.

[4]Russell N J,Gacesa P.Chemistry and biology of the mucoid strains of Pseudomonas aeruginosa in cystic fibrosis[J].Molecular aspects of medicine,1988,10(1):1-91.

[5]Linker,A,Jones R S.A polysaccharide resembling alginic acid from a Pseudomonas microorganism[J].Nature,1964,204(4954):187-188.

[6]Papageorgiou S K,Katsaros F K,Kouvelos E P,et al.Heavy metal sorption by calcium alginate beads from Laminaria digitata[J].Journal of Hazardous Materials,2006,137(3):1765-1772.

[7]Ensor S A,Sofos JN,Schmidt G R.Optimization of algin/calcium binder in restructured beef[J].Journal of Muscle Foods,1990,1(3):197-206.

[8]Leung V,Hartwell R,Elizei S S,et al.Postelectrospinning modifications for alginate nanofiber-based wound dressings[J].Journal of Biomedical Materia research Part B,2014,102(3):508-515.

[9]Wang Q,Zhang N,Hu X,et al.Alginate/polyethlene glycol blend fibers and their properties for drug controlled release[J].Journal of biomedical materials research Part A,2007,82(1):122-128.

[10]Iwamoto Y,Xu X,Tamura T,et al.Enzymatically depolymerized alginate oligomers that cause sytotoxic cytokine production in human mononuclear cells[J].Bioscience,biotechnology,and biochemistry,2003,67(2):258-263.

[11]Hu J,Geng M,Li J,et al.Acidic oligosaccharide sugar chain,a marine-derived acidic oligosaccharide,inhibits the cytotoxicity and aggregation of amyloid beta protein[J].Journal of pharmacological sciences,2004,95(2):248-255.

[12]Fujimura T,Kawai T,Kajiwara T,et al.Protoplast isolation in the marine brown alga Dictyopteris prolifera,(Dictyotales)[J].Plant Cell Reports,1995,14(9):571.

[13]Wong T Y,Preston L A,Schiller N L.Alginate lyase:review of major sources and enzyme characteristics,structure-function analysis,biological roles,and applications[J].Annual Reviews in Microbiology,2000,54(1):289-340.

[14] Lipoza, Waishi, Jiang dao, et al, research on seaweed tool enzyme-alginate lyase, advanced bioengineering, 2011,27(6): 838-.

[15]Han W,Gu J,Cheng Y,et al.Novel alginate lyase(Aly5)from a polysaccharode-degrading marine bacterium,Flammeovirga sp.MY04:effects of module truncation on biochemical characteristics,alginate degradation patterns,and oligosaccharide-yielding properties[J].Appl.Environ.Microbiol.,2016,82(1):364-374.

[16] Korea, quality of pass, Wangdan, etc. algin lyase is used in preparation of series oligosaccharide products, China, 201710651814[ P ].2018.01.12.

[17]Cheng Y,Wang D,Gu J,et al.Biochemical characteristics and variable alginate-degrading modes ofa novel bifunctional endolytic alginate lyase[J].Appl.Environ.Microbiol.,2017,83(23):e01608-17.

[18]Peng C,Wang Q,Lu D,et al.A novel bifunctional endolytic alginate lyase with variable action modes and versatile monosaccharide-yielding properties[J].Frontiers in Microbiology,2018,9:167.

[19] Korean Jun, Cheng Yuan May Yuan, Li Jun Pigeon, etc., an M-oriented monosaccharide excision type algin lyase Aly6, its coding gene and application, China, 201810887656[ P ].2018.12.21.

[20] Korean monarch, Zengliang albizzia, Gujing Yan, etc. one kind of broad spectrum polysaccharide degrading enzyme rAly16-1 from Streptomyces and its coding gene and application, China, 202110379188P 2021.04.09.

SEQUENCE LISTING

<110> Shandong university

<120> Achromobacter persicae JZB09, alginate lyase rAly06925, and coding gene and application of the enzyme

<160> 68

<170> PatentIn version 3.5

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atgttttttc tatcctttat aggtttaatt tcctgttcat cggtgagtca aaagcaaaaa 60

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caaacaccat ttgctgttac gcataaaacg ggtgttccac caagcggaga tcagcatgat 240

tacctgtcaa tagcgcctta tttctggcct gacgcttcga caaataatgg tttgccttat 300

gtcagaaaag acggagagat aaatccggag gcaagaaata accatacaga ctttaatgag 360

ctaattgctt tttttgatgc tatagcgacc ttaagagatg cctatttttt ttcggaggaa 420

atagtgtttg cggaaaaggc cctggagttg attagcgtct ggtttttgga ggcaagtact 480

aaaatgaatc caaaccttaa ttttggccag ggtataccgg gaaaaattga gggtcggtgt 540

tttggtatta ttgaatttga tcgcatcaca gaggtactaa aatgtctgga gcagtttaaa 600

aaaacaggag tactaccgat aaacatcgaa aatggaatga atcaatggct tgcatcttac 660

gcttcctggc ttcagcacag taggctgggg gtggaagaat ctaccagatt aaataatcat 720

ggtacccatt atgatgtgca gttattaagt attttaacct acctgggtag acttgatgaa 780

gttcgcatgc acctgaatac ggtcaccaaa agccggatat ttagccaaat agagccggat 840

ggtagtcagc caagggaact tgagcgaacg aaatcattct cctattcggt gatgaattta 900

catggttttt taaaattgtc tgagataggg aaaaaagttg gtgtgaaggt ttggaggatg 960

gaatcagaag atgggagaag tataaagaag ggctaccttt atttacttcc ttatttgacc 1020

ggaacgcaaa aatgggagca cagacagatt aaatcagtgg atcaaagtat tgaaaagctg 1080

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Met Phe Phe Leu Ser Phe Ile Gly Leu Ile Ser Cys Ser Ser Val Ser

1 5 10 15

Gln Lys Gln Lys Thr Ile Ser Leu Thr Asp Tyr Asp Ala Leu Lys His

20 25 30

Ala Lys Ala Leu Leu Ala Lys Asn Asp Pro Lys Val Lys Glu Asp Tyr

35 40 45

Ala Pro Leu Phe Ile Lys Ala Lys Ala Leu Leu Asn Gln Thr Pro Phe

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Ala Val Thr His Lys Thr Gly Val Pro Pro Ser Gly Asp Gln His Asp

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Tyr Leu Ser Ile Ala Pro Tyr Phe Trp Pro Asp Ala Ser Thr Asn Asn

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Gly Leu Pro Tyr Val Arg Lys Asp Gly Glu Ile Asn Pro Glu Ala Arg

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Asn Asn His Thr Asp Phe Asn Glu Leu Ile Ala Phe Phe Asp Ala Ile

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Ala Thr Leu Arg Asp Ala Tyr Phe Phe Ser Glu Glu Ile Val Phe Ala

130 135 140

Glu Lys Ala Leu Glu Leu Ile Ser Val Trp Phe Leu Glu Ala Ser Thr

145 150 155 160

Lys Met Asn Pro Asn Leu Asn Phe Gly Gln Gly Ile Pro Gly Lys Ile

165 170 175

Glu Gly Arg Cys Phe Gly Ile Ile Glu Phe Asp Arg Ile Thr Glu Val

180 185 190

Leu Lys Cys Leu Glu Gln Phe Lys Lys Thr Gly Val Leu Pro Ile Asn

195 200 205

Ile Glu Asn Gly Met Asn Gln Trp Leu Ala Ser Tyr Ala Ser Trp Leu

210 215 220

Gln His Ser Arg Leu Gly Val Glu Glu Ser Thr Arg Leu Asn Asn His

225 230 235 240

Gly Thr His Tyr Asp Val Gln Leu Leu Ser Ile Leu Thr Tyr Leu Gly

245 250 255

Arg Leu Asp Glu Val Arg Met His Leu Asn Thr Val Thr Lys Ser Arg

260 265 270

Ile Phe Ser Gln Ile Glu Pro Asp Gly Ser Gln Pro Arg Glu Leu Glu

275 280 285

Arg Thr Lys Ser Phe Ser Tyr Ser Val Met Asn Leu His Gly Phe Leu

290 295 300

Lys Leu Ser Glu Ile Gly Lys Lys Val Gly Val Lys Val Trp Arg Met

305 310 315 320

Glu Ser Glu Asp Gly Arg Ser Ile Lys Lys Gly Tyr Leu Tyr Leu Leu

325 330 335

Pro Tyr Leu Thr Gly Thr Gln Lys Trp Glu His Arg Gln Ile Lys Ser

340 345 350

Val Asp Gln Ser Ile Glu Lys Leu Val Asn Asp Leu Val Phe Ala Tyr

355 360 365

Arg Phe Phe Glu Ala Glu Glu Phe Ala Ser Val Ala Asp Ala Ala Lys

370 375 380

His Gly Asp Phe Phe Trp Gly Asn Phe Val Glu Ala Phe Phe Gln

385 390 395

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<212> DNA

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aaaggggagc cgcatcctcc ttcactttcg 30

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<210> 12

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ccttaatcgc aaggggagca taatcctcc 29

<210> 13

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<400> 13

ctttttgcga aggctaaagc attattaaat c 31

<210> 14

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<400> 14

agccttcgca aaaaggggag cataatcctc 30

<210> 15

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gactttaatg gcgtaattgc tttttttgat g 31

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tacgccatta aagtctgtat ggttatttc 29

<210> 17

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ataacgcgac agactttaat gagctaattg c 31

<210> 18

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aagtctgtcg cgttatttct tgcctccgg 29

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cagacgcgaa tgagctaatt gctttttttg 30

<210> 20

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gctcattcgc gtctgtatgg ttatttcttg 30

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actttgcgga gctaattgct ttttttgatg 30

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ttagctccgc aaagtctgta tggttatttc 30

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<400> 23

ttttggcgcg ggtataccgg gaaaaattga g 31

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<400> 24

gtatacccgc gccaaaatta aggtttggat tc 32

<210> 25

<211> 31

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aaataatgcg ggtacccatt atgatgtgca g 31

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ggtacccgca ttatttaatc tggtagattc 30

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tggtaccgcg tatgatgtgc agttattaag 30

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tcatacgcgg taccatgatt atttaatctg 30

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tggtacccat gcggatgtgc agttattaag 30

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atccgcatgg gtaccatgat tatttaatct g 31

<210> 31

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tcattctccg cgtcggtgat gaatttacat g 31

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cgacgcggag aatgatttcg ttcgctcaag 30

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tggtacccat gaggatgtgc agttattaag 30

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atcctcatgg gtaccatgat tatttaatct g 31

<210> 35

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tggtacccat caggatgtgc agttattaag 30

<210> 36

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atcgtcatgg gtaccatgat tatttaatct g 31

<210> 37

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tggtacccat gatgatgtgc agttattaag 30

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atcatcatgg gtaccatgat tatttaatct g 31

<210> 39

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tggtacccat aacgatgtgc agttattaag 30

<210> 40

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atcttgatgg gtaccatgat tatttaatct g 31

<210> 41

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tggtacccat catgatgtgc agttattaag 30

<210> 42

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<400> 42

atcgtaatgg gtaccatgat tatttaatct g 31

<210> 43

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<400> 43

tggtacccat aaggatgtgc agttattaag 30

<210> 44

<211> 31

<212> DNA

<213> Artificial sequence

<400> 44

atcttcatgg gtaccatgat tatttaatct g 31

<210> 45

<211> 30

<212> DNA

<213> Artificial sequence

<400> 45

tggtacccat cgagatgtgc agttattaag 30

<210> 46

<211> 31

<212> DNA

<213> Artificial sequence

<400> 46

atcgctatgg gtaccatgat tatttaatct g 31

<210> 47

<211> 30

<212> DNA

<213> Artificial sequence

<400> 47

tggtacccat tccgatgtgc agttattaag 30

<210> 48

<211> 31

<212> DNA

<213> Artificial sequence

<400> 48

atcggaatgg gtaccatgat tatttaatct g 31

<210> 49

<211> 30

<212> DNA

<213> Artificial sequence

<400> 49

tggtacccat ggggatgtgc agttattaag 30

<210> 50

<211> 31

<212> DNA

<213> Artificial sequence

<400> 50

atccccatgg gtaccatgat tatttaatct g 31

<210> 51

<211> 30

<212> DNA

<213> Artificial sequence

<400> 51

tggtacccat gtcgatgtgc agttattaag 30

<210> 52

<211> 31

<212> DNA

<213> Artificial sequence

<400> 52

atccagatgg gtaccatgat tatttaatct g 31

<210> 53

<211> 30

<212> DNA

<213> Artificial sequence

<400> 53

tggtacccat ttggatgtgc agttattaag 30

<210> 54

<211> 31

<212> DNA

<213> Artificial sequence

<400> 54

atcaacatgg gtaccatgat tatttaatct g 31

<210> 55

<211> 30

<212> DNA

<213> Artificial sequence

<400> 55

tggtacccat atcgatgtgc agttattaag 30

<210> 56

<211> 31

<212> DNA

<213> Artificial sequence

<400> 56

atctagatgg gtaccatgat tatttaatct g 31

<210> 57

<211> 30

<212> DNA

<213> Artificial sequence

<400> 57

tggtacccat ttcgatgtgc agttattaag 30

<210> 58

<211> 31

<212> DNA

<213> Artificial sequence

<400> 58

atcaagatgg gtaccatgat tatttaatct g 31

<210> 59

<211> 30

<212> DNA

<213> Artificial sequence

<400> 59

tggtacccat ccagatgtgc agttattaag 30

<210> 60

<211> 31

<212> DNA

<213> Artificial sequence

<400> 60

atcggtatgg gtaccatgat tatttaatct g 31

<210> 61

<211> 30

<212> DNA

<213> Artificial sequence

<400> 61

tggtacccat tgggatgtgc agttattaag 30

<210> 62

<211> 31

<212> DNA

<213> Artificial sequence

<400> 62

atcaccatgg gtaccatgat tatttaatct g 31

<210> 63

<211> 30

<212> DNA

<213> Artificial sequence

<400> 63

tggtacccat atggatgtgc agttattaag 30

<210> 64

<211> 31

<212> DNA

<213> Artificial sequence

<400> 64

atctacatgg gtaccatgat tatttaatct g 31

<210> 65

<211> 30

<212> DNA

<213> Artificial sequence

<400> 65

tggtacccat tgcgatgtgc agttattaag 30

<210> 66

<211> 31

<212> DNA

<213> Artificial sequence

<400> 66

atcacgatgg gtaccatgat tatttaatct g 31

<210> 67

<211> 30

<212> DNA

<213> Artificial sequence

<400> 67

tggtacccat tccgatgtgc agttattaag 30

<210> 68

<211> 31

<212> DNA

<213> Artificial sequence

<400> 68

atcaggatgg gtaccatgat tatttaatct g 31