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Heat resistance cutinase and its coding gene and expression

A technology for encoding genes and cutinase, applied in genetic engineering, plant genetic improvement, hydrolase and other directions, can solve the problems of low enzyme production by wild fungi, less research on cutinase, and no industrial production of cutinase.

Active Publication Date: 2008-04-30
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Domestic research on cutinase is less, the enzyme production of wild bacteria is low, and there is no case of industrial production of cutinase

Method used

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  • Heat resistance cutinase and its coding gene and expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 This example illustrates the purification procedure of cutinase.

[0044] Using Thermobifida fuscaWSH03-11 as the starting strain, in the seed medium (soluble starch 20g / L, beef extract 10g / L, yeast extract 5g / L, K 2 HPO 4 2g / L, NaCl 5g / L, pH8.0) 50℃, 200rpm for 20 hours, and then inoculated into the fermentation medium (sodium acetate 7.5g / L, yeast extract 7.5g / L, peptone 5g / L, K 2 HPO 4 2g / L, NaCl 5g / L, keratin 1g / L, pH8.0), 50°C, 200rpm culture for 50h, the cutinase fermentation broth was centrifuged at 4°C, 10000rpm for 20min to remove bacteria. The supernatant was collected through an activated carbon column. Add 70% solid ammonium sulfate to the clear solution passed through the activated carbon column overnight, centrifuge at 4°C and 10,000 rpm for 20 minutes, take the precipitate and dissolve it in buffer A (20mM Tris-HCl, pH 8), add 20% ammonium sulfate, After 0.22μm membrane filtration, the loaded sample is made. After the Phenyl HP hydrophobic column i...

Embodiment 2

[0045] Example 2 This example illustrates the identification process of the cutinase gene.

[0046] The obtained pure cutinase was sequenced by peptide fingerprinting. The sequencing results showed that it was very similar to the protein Tfu_0883 encoded by Thermobifida fusca in the NCBI database. The N-terminal sequence was ANPYERGP, which was the same as the protein Tfu_0883. The protein Tfu_0883 was predicted to be triglyceride by sequence , No indication of its cutinase activity. The pure wild fungus cutinase is subjected to enzymatic hydrolysis of cutin (apple peel treated with chloroform and methanol, pectinase and cellulase). Add 300 mg of cutin to pH 8.0, 10 mL of 50 mM potassium phosphate buffer, add purified cutinase, and shake at 30°C for 18 hours. After the reaction is completed, add glacial acetic acid to terminate the reaction. The fatty acid product is extracted with chloroform and methylesterified and silanized. -MS measurement revealed that a large amount of kerat...

Embodiment 3

[0047] Example 3 This example illustrates the isolation and cloning procedure of the cutinase encoding gene.

[0048] The Thermobifida fusca strain was cultured in LB liquid medium (peptone 10g / L, yeast extract 5g / L, NaCl10g / L) for 2 days, centrifuged at 10000rpm to collect the bacteria, washed with sterile water, and collected the precipitate and suspended in 500μL Tris-EDTA (three Hydroxymethylaminomethane-ethylenediaminetetraacetic acid) buffer, add 15μL of lysozyme, incubate at 37℃ for 30min, then add 5μL of RNase, incubate at 37℃ for 30min, add 30μL of 10% SDS (sodium dodecyl sulfate) ) And 15μL of proteinase K, incubate at 37℃ for 60min, add 100μL of NaCl (5M) and 80μL CTAB (hexadecyltrimethylammonium bromide), incubate at 65℃ for 20min, use 700μL of phenol: chloroform: isoamyl alcohol (25:24:1) extraction, centrifugation at 10000rpm, the supernatant was extracted with 700μL of chloroform:isoamyl alcohol (24:1), centrifuged at 10000rpm, the supernatant was mixed with 1400μL ...

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Abstract

A heat-resistant cutinase and its coding gene and expression belong to the technical field of bioengineering. The present invention provides a bacterial cutinase different from fungal cutinase, its encoding gene and expression system, including extracting the total DNA of Thermobifida fusca WSH03-11, designing primers, and PCR amplification to obtain the gene encoding heat-resistant cutinase, It has the nucleotide sequence shown in SEQ ID NO: 1, the full length is 906 nucleotides, and it encodes 301 amino acids. The plasmid pET20b(+) is used as the expression vector, and E.coli BL21 Rosetta(DE3)PlysS is used as the expression vector. The host can realize the high-efficiency expression of the heat-resistant cutinase gene. This cutinase has good thermal stability and has a good hydrolysis effect on cutin, triglyceride and various soluble synthetic esters, and can be widely used in textile, detergent and other industries.

Description

Technical field [0001] The invention relates to a heat-resistant cutinase and its coding gene and expression, belonging to the technical field of bioengineering. Background technique [0002] Cutinase is a hydrolase capable of hydrolyzing keratin macromolecules. As a multifunctional enzyme, it is widely used in many fields such as textile industry, food industry and chemical industry. [0003] (1) Application in biocatalysis [0004] In industrial products and processes, cutinase has unique application advantages. From soluble synthetic esters to insoluble long-chain triglycerides of various esters, cutinase has hydrolysis activity for them, and cutinase can carry out the transesterification of fats and oils or alcohols (stereotypes) under low water activity. ) Selective esterification; it can carry out the esterification and transesterification reaction between butyric acid and 2-butanol, methyl-, ethyl-, propyl propionate and other substances, and can also participate in the sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N1/21
Inventor 陈坚吴敬陈晟
Owner JIANGNAN UNIV
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