Novel cold-adapted alginate lyase AlgA7 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 consumption in the process of industrial application. It is of great significance to study the cold-adapted alginate lyase that can function and study its degradation conditions and final products

Method used

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  • Novel cold-adapted alginate lyase AlgA7 and application thereof
  • Novel cold-adapted alginate lyase AlgA7 and application thereof
  • Novel cold-adapted alginate lyase AlgA7 and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Artificial Design and Sequence Analysis of Alginate Lyase AlgA7

[0032] Alginate lyase gene of the present invention algA7 Artificially designed, fully synthetic sequence (synthesized by Huada Gene Company), including 1,578 base sequences, encoding 526 amino acid sequences, and its N-terminal contains a carbohydrate binding domain (Met 1 -Pro 176 ). Its C-terminus contains a conserved catalytic region of alginate lyase (Ser 177 -Asp 526 ), the binding domain can form a stable tertiary structure, which affects the thermal stability and thermal recovery of the enzyme. Using the National Center for Biotechnology Information (NCBI ) Conserved domain analysis in Conserved Domain (CDD) and multiple Sequence Alignment The C-terminal of the sequence contains a conserved region of the Alginate lyase 2 superfamily, and the N-terminal contains a carbohydrate binding domain. Multiple Sequence Alignment Basic The Local Alignment Search Tool (Blast) found that...

Embodiment 2

[0033] Example 2 Gene Cloning and Recombinant Expression of Alginate Lyase AlgA7

[0034] Fully synthesized in Example 1 algA7 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: PAlgA7EF:

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

[0037] Reverse primer: SEQ ID NO.4: PAlgA7ER:

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

[0039] The specific PCR amplification conditions are: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, a total of 30 cycles; extension at 72°C for 5 minutes; stabilization at 4...

Embodiment 3

[0042] Embodiment 3 Fermentation process and purification preparation method of alginate lyase AlgA7

[0043] The Escherichia coli BL21(DE3) / pET22b-AlgA7 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; 50 μg / mL ampicillin in LB liquid medium (500 mL Erlenmeyer flask loaded with 50 mL liquid medium), shake culture at 180 rpm at 37°C until OD 600 =0.6. The 5 L fermenter was loaded with 3 L of Terrific Broth (TB) medium, and sterilized in advance; 50 μg / mL ampicillin was added to the fermenter, and the cultured bacterial solution in the Erlenmeyer flask was 2% The inoculum was inoculated into a 5 L fermenter. 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 =5.0, add the inducer isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0...

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Abstract

The invention relates to novel alginate lyase with cold-adapted characteristics and an application thereof. The alginate lyase is artificially-designed novel alginate lyase AlgA7y, an amino acid sequence is as shown by SEQ ID NO.1, an N-end N comprises a section of carbohydrate combination domain (Met1-Pro176), an C-end C comprises a section of alginate lyase preserved catalytic area (Ser177-Asp526), and the identity between the amino acid sequence of the alginate lyase in provided bythe invention and the alginate lyase with known functions is only 71 percent. The alginate lyase AlgA7 of provided by the invention has cold-adapted characteristics, the most appropriate reaction temperature is 25 DEG C, and the reaction activity at 20 to and 10 DEG C can reach 81.9 percent to and42.6 percentof the reaction activity at the most appropriate temperature. The alginate lyase can control the enzymatic reaction to be performed at a low temperature, so that the energy consumption in the industrialized application process can be saved, and the cost can be savedreduced.

Description

technical field [0001] The invention relates to alginate lyase AlgA7 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 oligosaccharides ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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