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Novel gene of esterase and recombinant expression system

An expression vector, esterase technology, applied in genetic engineering, plant genetic improvement, recombinant DNA technology, etc.

Inactive Publication Date: 2009-04-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The current energy crisis has attracted people's attention. Traditional fossil energy can no longer meet human needs. The technology of synthesizing biodiesel through lipase biocatalysis has the advantages of simple extraction, mild reaction conditions, small amount of alcohol, easy recovery of glycerin and no waste generation. , no pollution and other advantages

Method used

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  • Novel gene of esterase and recombinant expression system
  • Novel gene of esterase and recombinant expression system
  • Novel gene of esterase and recombinant expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Obtaining of est_p1 gene

[0024] 1. Preliminary screening of the library: Dilute and spread the strains in the library on an LB plate containing 1% tributyrin, incubate at 37°C, pick out the colony that produces a transparent circle after 1 to 2 days;

[0025] 2. Re-screening of the library: Spot the picked strains on an LB plate containing 1% tributyrin, incubate at 37°C, confirm and select colonies that produce transparent circles after 1-2 days.

[0026] 3. Pick the strain that produces the transparent circle for liquid culture, extract the plasmid with the TIANGEN plasmid extraction kit, and send it to the company for sequencing;

[0027] 4. Sequencing results: the size of the inserted fragment of the foreign DNA of the plasmid is 3650bp. Four complete ORF sequences were predicted by ORF Finder. Among them, the gene est_p1 encoding esterase or lipase is located at 728-1618 bp of the inserted fragment. The size of est_p1 is 891bp, and its nucleotide seq...

Embodiment 2

[0044] Example 2 Construction of Est_p1 expression system and investigation of its enzymatic activity

[0045] 1. Construction of Est_p1 expression system

[0046] Designed to introduce NcoI and XhoI restriction sites at the 5' end and 3' end of Est_p1 respectively, delivered to Shanghai Sangong for synthesis, and used to construct expression vectors after correct sequencing. The synthetic est_p1 gene was double-digested with NcoI and XhoI; the pET28a (Novagen) vector was double-digested with NcoI and SalI, and then the above fragments were ligated with DNA ligase, and finally the ligated product was transformed into E. coli BL21 (DE3) In LB (containing kan 50μg / ml) plates, the positive clones were initially screened, and the pET28a-Est_p1 / BL21 recombinant expression system was obtained after verification by enzyme digestion.

[0047] 2. Induced expression and purification of target protein;

[0048] In this example, Ni-agarose affinity chromatography was used to purify the ...

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Abstract

The invention provides a new esterase gene named Est_p1 and a recombination expression system thereof. The gene is cloned from the metagenomic library of the marginal mud 100 meters under the South China Sea. The gene is an 891bp long amino acid numbered 296, which successfully constructs a recombination expression system of a colon bacillus named pET28a-Est_p1 / BL21. The molecular weight of a target protein is 33.5 kD. With pNP-butyric ester as catalyzing substrates, the Est_p1 is proved to be a mildly alkaline medium temperature esterase. The Est_p1 has strong catalytic activity for short esters and is applicable to esterification and transesterification industries.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to gene cloning and expression of non-culturable microorganisms. Background technique [0002] The huge amount of non-cultivable microorganisms contains rich resource advantages, and the study of metagenomics provides a reliable technical guarantee for mining this resource advantage. 95% of marine microorganisms cannot or are difficult to cultivate artificially under the existing experimental conditions, extract DNA directly from the environment, and establish a metagenomic library of genetic information that can theoretically cover all microorganisms in the environment. It is possible to obtain new genes with application value by heterologously expressing the gene fragments obtained in the extreme environment with a depth of more than 100 meters in the ocean. By constructing a marine sediment metagenomic library, the genetic diversity of marine microorganisms is preserved to facilitat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/63C12N1/21C12R1/19
Inventor 李颖彭晴关国华王珍芳李季伦
Owner CHINA AGRI UNIV
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