Method for preparing heat-resistant cutinase-CBD and application thereof in cotton fiber refining

A cutinase, cotton fiber technology, applied in the field of enzyme genetic engineering and enzyme engineering, can solve the problems of increasing the binding ability of enzymes to cotton fibers, etc., and achieve the effect of high-efficiency expression

Active Publication Date: 2009-12-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fusion of CBD and scouring enzymes (such as cutinase) can increase the binding ability of the enzyme to cotton fibers, so as to better hydrolyze the impuritie

Method used

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  • Method for preparing heat-resistant cutinase-CBD and application thereof in cotton fiber refining
  • Method for preparing heat-resistant cutinase-CBD and application thereof in cotton fiber refining
  • Method for preparing heat-resistant cutinase-CBD and application thereof in cotton fiber refining

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: This example illustrates the extraction of total DNA from Thermobifida fusca WSH03-11.

[0040] Thermobifida fusca WSH03-11 strain was cultured in LB liquid medium (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L) at 50°C for 2 days, and 3mL of bacterial liquid was collected by centrifugation at 12000rpm. Genomic DNA extraction kit method to extract WSH03-11 total DNA;

Embodiment 2

[0041] Example 2: This example illustrates the cloning procedure of the thermostable cutinase-CBD encoding gene.

[0042] Two pairs of primers P1, P2 and P3, P4 were designed according to the gene sequences of Tfu_0883 and Tfu_1074 registered on NCBI. The underlines are restriction sites Nco I and EcoR I,

[0043] P1: 5'-GGAATACCATATGT CCATGG CCAACCCCTACGAGCGCGG-3'

[0044] P2: 5'-CGCGGCGATCGCCATGAACGGGCAGGTGGA-3'

[0045] P3: 5'-TCCACCTGCCCGTTCATGGCGATCGCCGCG-3'

[0046] P4: 5'-CATCTCGAGA GAATTC GGGCAGGTAAGGGTCGGAACAG-3'

[0047] Using pET20b-Tfu_0883 plasmid DNA as template and P1 and P2 as primers, cutinase gene was amplified by PCR. Using the total DNA of Thermobifida fusca WSH03-11 as template and P3 and P4 as primers, the gene of cellulase CBD was amplified by PCR. PCR reaction in 50μL system (refer to TaKaRa Ex Taq TM kit), the reaction conditions are 95°C denaturation for 5 minutes, followed by 1 minute denaturation at 95°C, 1 minute annealing at 55°C, and 1.5...

Embodiment 3

[0048] Example 3: This example illustrates the construction procedure of the thermostable cutinase-CBD gene in Escherichia coli expression vector.

[0049] The plasmid used to construct the expression vector in Escherichia coli is pET20b(+), with pelB signal peptide and His-tag marker. The pET20b(+) plasmid and the cutinase-CBD gene were digested with Nco I and EcoR1. After the digested product was recovered from rubber tapping, it was ligated with T4 ligase at 16°C overnight, and the ligated product was transformed into E.coli JM109 competent cells. Cultivate overnight at 37°C, select transformants for liquid culture with 100mg / L ampicillin LB, and then extract the plasmid to obtain the enriched Tfu_0883-CBD / pET20b(+) plasmid.

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Abstract

The invention discloses a method for preparing heat-resistant cutinase-CBD and application thereof to cotton fiber refining, which belong to the field of enzyme gene engineering. The method comprises the steps: taking pET20b-Tfu_0883 plasmid DNA and total DNA of thermophilic ascomycete WSH03-11 as a template, performing PCRs of designed primers respectively to obtain an encoding cutinase Tfu_0883 gene and a CBD gene in cellulose Tfu_1074, and then obtaining a fusion gene of encoding cutinase-cellulose-binding domain (CBD) (shortened as the heat-resistant cutinase-CBD) through overlap PCR; and taking pET20b(+) as an expression vector and E.coli BL21(DE3) as an expression host to realize the high level expression of a heat-resistant cutinase-CBD gene. The fusion enzyme has the activity of cutinase, the optimum temperature is 50 DEG C, the optimum pH value is 8, and the activity of the enzyme can still reach over 50 percent when heat preservation for 50h at a temperature of 50 DEG C. The cutinase-CBD has an absorption rate of 50 percent on cotton fiber, reaches reaction balance after 6h, and generates 180mu mol of fatty acid. The fusion enzyme is suitable for cotton fiber refining.

Description

technical field [0001] The invention relates to the preparation of a heat-resistant cutinase-CBD gene and its application in cotton fiber scouring, belonging to the fields of enzyme gene engineering and enzyme engineering. Background technique [0002] In the scouring process of cotton fabrics, in order to obtain excellent wettability and whiteness of cotton fibers, it is necessary to remove various companions such as cutin, cotton wax, pectin and protein that seriously affect the appearance, wearability and dyeing and finishing of cotton fabrics. biology. At present, the traditional strong alkali high-temperature treatment method has been used in China. It has many shortcomings, such as complicated and lengthy process, huge equipment, large fiber damage, high water consumption and energy consumption, and produces a large amount of sewage, which is extremely unfavorable to the ecological environment. As the society pays more and more attention to the environmental protectio...

Claims

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Application Information

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IPC IPC(8): C12N9/96C12N15/55C12N15/70D06M16/00C12R1/19D06M101/06
Inventor 吴敬陈坚张瑶陈晟
Owner JIANGNAN UNIV
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