Cold-adapted alginate lyase AlgA5 and application thereof

A technology of alginate lyase and cold adaptation, which is applied in the directions of lyase, carbon-oxygen lyase, application, etc., can solve the problems of increased energy consumption and low activity, and achieves the effect of reducing energy consumption and good industrial application prospect.

Inactive Publication Date: 2018-12-18
王存良
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the reported alginate lyases have very low activity at low temperature and must be heated to a certain degree (40-50°C) to function, which greatly increases the energy consum

Method used

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  • Cold-adapted alginate lyase AlgA5 and application thereof
  • Cold-adapted alginate lyase AlgA5 and application thereof
  • Cold-adapted alginate lyase AlgA5 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Artificial Design and Sequence Analysis of Alginate Lyase AlgA5

[0032] Alginate lyase gene of the present invention algA5 For artificial design, the fully synthetic sequence (synthesized by Huada Gene Company) contains 1,107 base sequences, encoding 369 amino acid sequences, and a F5-F8-Type C domain containing 61 amino acids is designed in the sequence ( Glu 160 -Gly 220 ), the binding domain can form a stable tertiary structure, which affects the cold adaptability of the enzyme. Using the National Center for Biotechnology Information (NCBI )middle Conserved domain analysis Conserved domain (CDD) found that, The sequence contains a conserved region of the Alginate lyase 2 superfamily. Multiple sequence comparison Basic Local AlignmentSearch Tool (Blast) analysis found that the highest amino acid sequence similarity with AlgA5 is the polysaccharide lyase family 7 (PL-7) alginate lyase and AlgA5 have the highest amino acid sequence similarity of 75% ...

Embodiment 2

[0033] Example 2 Gene cloning and recombinant expression of alginate lyase AlgA5

[0034] Fully synthesized in Example 1 algA5 restriction endonuclease Nco I and xho I (purchased from Dalian Bao Biological Co., Ltd.) is the enzyme cutting site and the protection base of the enzyme cutting site is designed, and the recombinant primers are designed as follows (the underline is the restriction endonuclease site, and the italic is the restriction endonuclease protection base) :

[0035] Forward primer: SEQ ID NO.3: PAlgA5EF:

[0036] 5'- CATG CCATGG GTGGCGCCCTCGCCTCCGAAGCCCCCCGC-3' ( Nco I)

[0037] Reverse primer: SEQ ID NO.4: PAlgA5ER:

[0038] 5'- CCG CTCGAG GCCGTGGGTTTCGCCGACGCGG-3' ( xho I)

[0039] The above-mentioned recombinant primers were used to PCR amplify the alginate lyase gene, and the high-fidelity DNA polymerase Primerstar HS used in PCR was purchased from Dalian Bao Biology Company. The specific PCR amplification conditions are: pre-dena...

Embodiment 3

[0043] Example 3 Fermentation process and purification preparation method of alginate lyase AlgA5

[0044] The Escherichia coli BL21(DE3) / pET22b-AlgA5 constructed and stored at -80°C in Example 2 was streaked on the LB solid plate, and after culturing at 37°C for 16 hours, single clones were picked; the single clones were transferred to cells containing 50 μg / mL ampicillin in LB liquid medium (500 mL Erlenmeyer flask loaded with 50 mL liquid medium), cultured in a shaker at 37°C at 180 rpm to OD 600 =0.6. The 5 L fermenter was loaded with 60% (3 L) Terrific Broth (TB) medium, and sterilized in advance; 50 μg / mL ampicillin was added to the fermenter, and the cultured bacteria in the Erlenmeyer flask The solution was inoculated into a 5 L fermenter according to the inoculum amount of 2%. Adjust the initial ventilation rate to 50 L / h, the initial rotation speed to 350 rpm, the temperature at 37°C, and the dissolved oxygen at 15-40%; when the bacteria grow to OD 600 When = 2.0, ...

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Abstract

The invention relates to alginate lyase with cold-adapted characteristics and an application thereof. The alginate lyase is a novel alginate lyase AlgA5, and an amino acid sequence is as shown by SEQID NO.1. In the sequence, a section of F5-F8-Type C structural domain (Gglu160-Ggly220) is designed, and the combinational structural domain can form a section of stable three-level structure, therebyinfluencing the cold-adapted performance of the lyase. The identity between the amino acid sequence of the alginate lyase AlgA5 and the sequence of the existing alginate lyase reaches up to 75 percent. The alginate lyase AlgA5 provided byof the invention has the cold-adapted characteristics, an acting way is an endonuclease way, a main product of the degradation of the cold-adapted alginate lyaseis unsaturatedalginate disaccharide and unsaturated alginate trisaccharide. The alginate lyase provided byof the invention is high in yield, stable in performance and high in industrialized application potential.

Description

technical field [0001] The invention relates to an endo-type alginate lyase AlgA5 with cold adaptability and application thereof, belonging to the field of biotechnology. Background technique [0002] Alginate is an important component of brown algae cell wall. , A linear polysaccharide linked by 4 glycosidic bonds. Generally, alginate is processed from large brown algae such as kelp, sargassum, and macroalgae. my country is a big country in seaweed farming, and the production of alginate accounts for more than 70% of the world's total production. Alginate is widely used in chemical, pharmaceutical, food and other industries. Alginate oligosaccharides with different degrees of polymerization have different biological activities. The latest research shows that GV-971, a poly-M segment oligosaccharide drug prepared from alginate, can inhibit the aggregation and cytotoxicity of β-amyloid cells with multiple targets. Phase III clinical trials have been completed; poly-G olig...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12P19/00C12P19/12
CPCC12N9/88C12P19/00C12P19/12C12Y402/02003
Inventor 王存良李尚勇
Owner 王存良
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