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A mutant of cutinase and its preparation method

A kind of cutinase and mutant technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of poor thermal stability of fungal cutinase, unsuitable for application, low hydrolysis efficiency and the like

Active Publication Date: 2011-11-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, fungal cutinase has poor thermal stability and is not suitable for application in the textile industry
And the heat-resistant cutinase produced by thermobifida fusca developed by our laboratory can effectively hydrolyze polyesters such as cutin and PET, but the hydrolysis efficiency is low (Chen Jian, Wu Jing, Chen Sheng. A kind of heat-resistant cutin Enzyme and its coding gene and expression. Application No. 200710026074.2)

Method used

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  • A mutant of cutinase and its preparation method
  • A mutant of cutinase and its preparation method
  • A mutant of cutinase and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Method for determining mutation sites of cutinase mutants of Thermobifida fusca.

[0026] Polyesters such as cutin are hydrophobic macromolecules composed of a large number of fatty acids. The size of amino acids around the active center of cutinase affects the combination of polyester macromolecules and active centers. In addition, the hydrophobicity of amino acids affects the substrate binding site and substrate. Therefore, based on the cutinase mimic structure, proceeding from the above two aspects, determine the mutation site and then perform site-directed mutation on the cutinase gene. Based on the analysis of the simulated structure of the cutinase of Thermobifida fusca, three sets of mutation series were designed: (1) The amino acid isoleucine I218 located directly above the active center of serine occupies a large space, which has a large impact on the active center and The combination of macromolecular substrates forms a large steric hindrance, so it...

Embodiment 2

[0027] Example 2: The preparation method of the cutinase mutant of Thermobifida fusca.

[0028] I218A-pet20b(+), W195 / F249-pet20b(+), Q132A / T101A-pet20b(+) mutant plasmids were constructed by rapid PCR site-directed mutagenesis.

[0029] Construction of I218A-pet20b(+), W195-pet20b(+), Q132A-pet20b(+) mutant plasmids using the CUT-pet20b(+) plasmid as a template (Chen Jian, Wu Jing, Chen Sheng. A heat-resistant cutinase And its coding gene and expression. Application No. 200710026074.2), use the mutation primers of I218A, W195A and Q132A to obtain the product with a size of about 4500bp by PCR respectively, transform the Escherichia coli JM109 competent cells after the PCR product is treated with DpnI, and select the transformants Sequencing verification.

[0030] The W195 / F249-pet20b(+), Q132A / T101A-pet20b(+) mutant plasmids were constructed using the verified correct W195-pet20b(+), Q132A-pet20b(+) plasmids as templates, and F249A and T101A as mutation primers , PCR amplif...

Embodiment 3

[0054] Example 3: Expression and purification method of cutinase mutants from Themobifida fusca.

[0055] The mutant plasmids I218A-pet20b(+), W195 / F249-pet20b(+), Q132A / T101A-pet20b(+) were transformed into E. coli BL21(DE3) cells, and the transformants were selected for sequencing verification. The positive transformant after verification was in TB medium (glycerol 5g / L, peptone 12g / L, yeast extract 24g / L, K 2 HPO 4 12.54g / L, KH 2 PO 4 2.31g / L) in 37°C liquid culture overnight, then inserted into TB fermentation liquid medium and cultured at 37°C for 3 hours, then induced with 4 mg / L IPTG (isopropylthioβD-galactoside), cooled to 25°C and incubated at constant temperature for 80 hours .

[0056] The fermentation broth was centrifuged at 10,000 rpm for 20 minutes at 4°C to remove bacteria. The supernatant was collected through an activated carbon column. Add 70% solid ammonium sulfate to the clear solution passing through the activated carbon column for salting out overn...

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Abstract

The invention provides a mutant of themobifida fusca cutinase and a preparation method thereof. The mutant comprises one or more substituents adjacent to amino acid residues corresponding to the active center of cutinase of thermobifida fusca. I218A, Q132A / T101A and W195A / F249A can improve catalyzing efficiency to cotton fiber. The catalyzing efficiency of mutant I218A to keratin is improved by 50% compared with that of the original cutinase. The catalyzing efficiency of Q132A / T101A on synthetic fibre ethylene glycol terephthalate (PET) is improved by 3 times compared with the original cutinase. The three types of mutants have wide application prospect in pre-treatment of textile fiber.

Description

technical field [0001] The present invention relates to an enzyme mutant and a preparation method thereof, in particular to a cutinase mutant and a preparation method thereof. A technique for the substrate specificity of thermocutinases. Background technique [0002] Cutinases are hydrolytic enzymes capable of hydrolyzing macromolecules of cutin. As a multifunctional enzyme, it has been widely used in many fields such as textile industry, food industry and chemical industry. Especially in the textile industry, cutinase can be used in cotton fiber bioscouring to remove wax and cutin on the surface of cotton fiber, and help to further remove impurities such as pectin and protein, so as to achieve the purpose of scouring. Studies in recent years have shown that cutinase can also be used to modify synthetic fiber polyethylene terephthalate (PET), increase fabric water absorption, improve hand feel, and improve fabric quality. Both of the above two processes currently adopt th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/70
Inventor 吴敬陈晟陈坚
Owner JIANGNAN UNIV
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