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A mutant of alginate lyase and its application

A technology of alginate lyase and mutants, which is applied in the field of alginate lyase mutants and its application, can solve the problems of poor substrate specificity and low enzyme activity, achieve strong pH stability and retain biological activity

Active Publication Date: 2020-12-25
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the currently screened alginate lyases have poor substrate specificity and low enzymatic activity, and there are very few alginate lyase products used in industrialization. Therefore, alginate cracking enzymes with high activity and wide substrate specificity are obtained. Enzymes have become the focus of current research, especially through genetic engineering and protein engineering to improve the catalytic activity of alginate lyase, which is favored by most researchers

Method used

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  • A mutant of alginate lyase and its application
  • A mutant of alginate lyase and its application
  • A mutant of alginate lyase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of alginate lyase mutant E226K

[0026] Alginate lyase gene in the present invention AlgL (The nucleotide sequence is shown in SEQ ID NO.1) derived from the seawater isolated from Zhoushan Islands Pseudoalteromonas sp. zb7-4, with a full length of 1203 bp, encodes 400 amino acids, the 1st-31st amino acid is a signal peptide, the 32-131st amino acid is a carbohydrate binding domain (i.e. the CBM domain), and the 197th-385th amino acid is a catalytic domain. Truncating between amino acids 157-158, truncating amino acids 1-157 to obtain alginate lyase truncation enzyme AlgL-T157N, the sequence of which is shown in SEQ ID NO.3.

[0027] Through sequence comparison, it was found that AlgL-T157N and 4Q8K have the same amino acid sequence in the catalytic domain, so the docking results were combined based on the research on the protein structure and catalytic mechanism of 4Q8K ( figure 1 ), using Discovery Studio2016 software to perform virtual ala...

Embodiment 2

[0035] Example 2 Induced expression and purification of truncation enzyme AlgL-T157N and mutant E226K

[0036] Extract the recombinant plasmid pET-22b(+)- AlgL-T157N and pET-22b(+)- E226K , transfer them into E. coli From BL21(DE3) competent cells, pick positive recombinants in 5 mL LB (Amp + ) liquid medium, cultured at 200 r / min at 37°C for 12 h, and then inoculated to 25 mL LB (Amp + ) culture medium, 37℃ 200 r / min culture OD 600 Between 0.6-0.8, IPTG (final concentration 0.2 mmol / L) was added to induce at 20°C 200 r / min for 24 h, and the activity of alginate lyase was determined. The LB medium without IPTG after the same inoculation was used as a blank control. The enzyme activity of mutant E226K was 7.14±0.09 U / mL, which was 1.11 times higher than that of AlgL-T157N (3.38±0.11 U / mL).

[0037] The activity of alginate lyase was determined by DNS method. Add 0.1 mL of enzyme solution to 0.9 mL of 0.3% sodium alginate substrate, incubate at 50°C for 15 min, add 1...

Embodiment 3

[0049] Example 3 Separation and purification of truncation enzyme AlgL-T157N and mutant E226K

[0050] (1) DEAE anion column purification: use AKTA purification system to purify protein, first use DEAE FF (HiTrap TM , 5 mL) prepacked column to purify the protein, the column was equilibrated with McIlvaine buffer with a concentration of 50 mmol / L and pH 7.0 at a flow rate of 5 mL / min, and the crude enzyme solution was filtered with a 0.22 μm filter membrane. Load the obtained filtrate onto the anion column, and then equilibrate the column at a flow rate of 2 mL / min. After equilibrating for 4-5 column volumes, use the pre-prepared 1 mol / L NaCl solution (50mmol / L, McIlvaine buffer solution with pH 7.0 Preparation) Gradient elution column, detect protein by UV detector, collect protein peaks under different gradient elution, detect enzyme activity, and perform SDS-PAGE to detect protein purity.

[0051] (2) Nickel column purification: upload the protein preliminarily purified by ...

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Abstract

The invention relates to an alginate lyase mutant and application, and belongs to the fields of enzyme engineering and gene engineering. According to the mutant, E on the 226th site is mutated to K based on the sequence SEQ ID NO.3, and through expression, the enzyme activity of the mutant is improved by 1.11 times, and compared with a truncated enzyme AlgL-T157N, the activity of crude enzyme obtained after being purified is improved by 1.03 times. The optimum temperature of the mutant is 55 DEG C, after the mutant is subjected to heat preservation at the pH of 6.0-8.0 for 1 hour, the enzyme activity is little changed, and the mutant is high in pH stability. Compared with the AlgL-T157N, the catalytic efficiencies (Kcat / Km) of the mutant to sodium alginate, Poly M and Poly G are improved by 10.22 times, 8.59 times and 2.97 times respectively. The method of combining structural sequence analysis with soft assist screening is adopted, the mutation site is determined, and through PCR site-directed mutagenesis, the mutant higher in catalytic activity is obtained, and a basis is laid for further industrial application.

Description

technical field [0001] The invention relates to a mutant of alginate lyase and application thereof, belonging to the fields of enzyme engineering and genetic engineering. Background technique [0002] Alginate lyase is a lyase that degrades alginate in brown algae. It mainly catalyzes the depolymerization of alginate through the β-elimination reaction of 4-O-glycosidic bonds, and at the non-reducing ends C-4 and C-5 An oligomeric uronic acid structure with unsaturated double bonds is produced between them. According to the substrate preference of alginate lyase, it can be divided into poly M, poly G and poly MG specific lyase; according to the different mode of action, it can be divided into endoenzyme and exoenzyme, endoenzyme mainly cuts seaweed The glycosidic bond inside the acid polymer produces unsaturated oligosaccharides, while the exonuclease further degrades the fucoidan oligosaccharides into monosaccharides. [0003] At present, with the deepening of research on ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12P19/12C12P19/02C12P19/00
CPCC12N9/88C12P19/00C12P19/02C12P19/12
Inventor 林娟曾德样许鑫琦
Owner FUZHOU UNIV
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