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Cutinase gene capable of efficiently producing cutinase and application thereof

A kind of cutinase and gene technology, applied in the field of cutinase gene, can solve problems such as high price, hindering the production of cutinase by recombinant bacteria, unsuitable for fermentation production and the like

Inactive Publication Date: 2013-05-08
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Most foreign scholars have studied the use of recombinant yeast strains to produce fungal cutinases. This method mainly has the following restrictive factors: (1) galactose-inducible promoters are mostly used in cutinase gene expression vectors, but as inducers, Galactose is also rapidly consumed as a carbon source during the fermentation process, so too fast consumption of galactose seriously hinders the ability of recombinant bacteria to produce cutinase; (2) In view of the above-mentioned rapid consumption of galactose, some scholars have proposed In the above, galactose is fed and fed to maintain the continued induction of enzyme production by the bacteria, but galactose is more expensive than glucose, glycerin, etc., and is not suitable for large-scale fermentation production in industry

Method used

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  • Cutinase gene capable of efficiently producing cutinase and application thereof
  • Cutinase gene capable of efficiently producing cutinase and application thereof
  • Cutinase gene capable of efficiently producing cutinase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 novel cutinase coding gene

[0023] A cutinase gene that can be used for high-efficiency production of cutinase, the nucleotide sequence of which is shown in SEQ ID NO.1. The method of obtaining it is to synthesize and optimize the gene encoding cutinase to obtain tfu, artificially add α-factor signal peptide and kozak sequence at the N-terminal, and clone it into pYES2.

Embodiment 2

[0024] Construction and identification of embodiment 2 recombinant yeast

[0025] Using Saccharomyces cerevisiae BY4741 as a gene template, four strong constitutive promoters: ADH1, HXT1, TEF1, and TDH3 were obtained according to the primers P1-P8 in Table 1, which were connected to the plasmid pYES2, and the inducible promoter GAL1 was used as a control to construct Four recombinant expression vectors were selected: pY-ADH1-tfu, pY-HXT1-tfu, pY-TEF1-tfu, pY-TDH3-tfu. The above four recombinant plasmids were transformed into S. cerevisiae INVsc1 competent cells by lithium acetate chemical method, and the colonies that could grow on the uracil-deficient YNB plate were successively transferred for three generations on the plate to obtain genetically stable recombinants. A number of positive recombinants were picked and verified by PCR with primers tfu-F and tfu-R, and a fragment of about 1095bp was obtained, indicating that the four recombinant plasmids had been successfully tra...

Embodiment 3

[0029] Example 3 Analysis and Comparison of Constitutive Promoter Reconstructed Saccharomyces Produced Cutinase

[0030] Inoculate the reconstituted strain into the seed medium, fill 50mL of a 500mL shaker flask with a temperature of 30°C, a rotational speed of 200r / min, and a culture time of 24-31h. According to the inoculum amount of 10%, the cultured seeds were inserted into the fermentation medium (initial sugar concentration was 20g / L), and cultivated under the conditions of 28°C and 200rpm. Compared with the control bacteria: TEF1p, TDH3p dry weight and enzyme All activities were improved; after 90 hours of fermentation, TEF1p had better dry weight and enzyme activity expression, which were 11.88U / mL and 17.85g·DCW / L, respectively. ADH1 had no effect, and the enzyme activity of HXT1 decreased instead.

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Abstract

The invention discloses a cutinase gene capable of efficiently producing cutinase and an application thereof, and particularly relates to an artificial encoding gene of the cutinase gene and an application thereof. The encoded cutinase gene is synthetically optimized to obtain tfu, and alpha-factor signal peptide and a kozark sequence are artificially added to an N end. Expression of the artificial cutinase gene in saccharomyces cerevisiae is successfully achieved by the invention. In addition, engineering bacteria with good enzyme production and performance are built by combining with the optimization of a promoter in a recombinant strain expression system; and a certain theoretical basis is established for industrial production of subsequent cutinase.

Description

technical field [0001] The invention relates to a cutinase gene which can be used for high-efficiency production of cutinase and its application, in particular to an artificial cutinase gene encoding gene. Background technique [0002] Cutinase is a hydrolytic enzyme that can degrade cutin and produce a large amount of fatty acid monomers. Cutinase has a wide range of sources and exists in plant pollen and various microorganisms. According to the source, cutinase can be divided into fungal cutinase, bacterial cutinase and pollen cutinase. Compared with fungal cutinase, bacterial cutinase has the advantages of high catalytic efficiency, strong environmental adaptability, and mature heterologous expression technology of genetic engineering. In the present invention, bacterial cutinase is selected as the target gene for expression. [0003] At present, Escherichia coli is mostly used as the expression host for domestic recombinant fermentation production of cutinase, which is...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/10C12N1/19C12N5/10C12N15/63C12N9/18C12R1/865
Inventor 王淼康振蔡彦秋陈坚堵国成
Owner JIANGNAN UNIV
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