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Recombinant alginate lyase and construction method and application thereof

A technology of alginate lyase and alginate, which is applied in the fields of application, hydrolase, botanical equipment and methods, etc., can solve the problems of low expression level and application of restriction enzymes, achieve high enzyme activity level, easy separation and purification, The effect of low impurity protein content

Active Publication Date: 2018-07-17
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, more than 20 alginate lyase genes have been cloned and sequenced, and a variety of recombinant alginate lyase genetic engineering bacteria have been constructed, but the expression level of recombinant alginate lyase gene engineering is low, which limits the application of the enzyme

Method used

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  • Recombinant alginate lyase and construction method and application thereof
  • Recombinant alginate lyase and construction method and application thereof
  • Recombinant alginate lyase and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Cloning and analysis of the full-length gene of alginate lyase Aly-Cob

[0029] (1) The gene encoding alginate lyase was obtained by constructing a Fosmid metagenomic library for the mixture of environmental samples and production waste in the kelp stacking place in the kelp production and processing plant. The gene was amplified by PCR. When designing primers, restriction sites BamH I and Hind III were added to the 5' ends of the primers respectively. The primer sequences were:

[0030] AlyC-F: 5'-CG GGATCC ATGCGTAACACCCGTGTGC-3', underlined is the BamH I restriction site;

[0031] AlyC-R: 5'-CCC AAGCTT TCATTGCATCTCACCGCTCT-3', the Hind III restriction site is underlined.

[0032] PCR conditions are: 94°C pre-denaturation, 3min; 94°C, 30s; 58°C, 30s; 72°C, 1min; a total of 30 cycles, the last 72°C, 10min. Agarose gel electrophoresis showed a specific band at about 1.0kb ( figure 1 ). It was excised from the agarose gel and purified using a DNA Gel Ex...

Embodiment 2

[0036] Embodiment 2: Recombinant bacterial construction and enzyme production

[0037] The construction of the recombinant strain E.coli BL21pLysS / pET-28a(+)-aly-cob: the target gene sequence aly-cob and the plasmid pET-28a(+) were cut with restriction endonucleases BamH I and Hind III respectively, and Transformed into E.coli BL21pLysS after T4 ligase ligation. Pick a single colony for liquid culture, extract the recombinant plasmid, verify it by PCR, and use BamH I and Hind III for double enzyme digestion verification. Get two fragments with lengths of about 5.3kb and 1kb respectively, such as figure 2 , proving that the alginate lyase gene has been successfully cloned into the expression vector pET-28a(+) and transformed into the expression host E.coli BL21pLysS, the recombinant plasmid was named pET-28a(+)-aly-cob, the recombinant strain Named as E.coliBL21pLysS / pET-28a(+)-aly-cob.

[0038] Induced expression of recombinant bacteria: insert the constructed recombinant ...

Embodiment 3

[0042] Example 3: Research on the enzymatic properties of recombinant alginate lyase Aly-Cob

[0043] The recombinant strain E.coli BL21pLysS / pET-28a(+)-aly-cob was induced to produce enzyme, and the enzymatic properties of the crude enzyme liquid obtained from fermentation were purified.

[0044] 1) Optimum temperature and temperature stability of recombinant alginate lyase Aly-Cob: Measure the enzyme activity of recombinant alginate lyase Aly-Cob solution at 25-65°C with appropriate concentration, respectively Each of the above-mentioned different temperatures was incubated for 30 minutes to measure the residual enzyme activity, and the highest enzyme activity was defined as 100%. In addition, the Aly-Cob enzyme solution was incubated in water baths at 35, 40, 45, 55, and 65°C, respectively. Samples were taken every 10 minutes to determine the residual enzyme activity of the sample, and the stability of the enzyme at different temperatures was determined. Finally, it was d...

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Abstract

The invention discloses a recombinant alginate lyase and a construction method and an application thereof, which belong to the technical field of biology. The alginate lyase Aly-Cob is derived from anenvironment sample (a mixture of sea-tangle stacking place and factory production waste material). By using a gene engineering technology, a gene of the alginate lyase is cloned to an expression vector, and a recombinant bacterial strain for heterogenous expression of the alginate lyase is obtained. The alginate lyase Aly-Cob produced by the bacterial strain through fermentation has the functionfor degrading sodium alginate to prepare alginate oligosaccharide. The provided alginate lyase Aly-Cob can be widely used for the fields of agriculture, food, feed addition, medicine and alga processing.

Description

technical field [0001] The invention relates to a recombinant alginate lyase and its construction method and application, belonging to the field of biotechnology. Background technique [0002] In recent years, with the vigorous development of marine drugs, the research on seaweed polysaccharides has been paid more and more attention, and alginate is one of them. Alginate has a wide range of application values. Due to its anti-tumor, immune regulation, hypoglycemic and blood lipid-lowering biological activities, alginate degradation products have become a representative direction and research hotspot for the in-depth research and development of high value-added utilization of alginic acid products in the world. Alginate lyase is used as a tool to enzymatically produce fucoidan oligosaccharides and low molecular weight polysaccharides. It has the advantages of mild degradation conditions, controllable process, and high yield. In addition, besides being able to prepare algal o...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/80C12P19/04
CPCC12N9/80C12P19/04
Inventor 史劲松许正宏李恒郝瑶龚劲松李会
Owner JIANGNAN UNIV
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