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Heat resistance cutinase and its coding gene and expression

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

Active Publication Date: 2012-01-25
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
  • Heat resistance cutinase and its coding gene and expression
  • 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 for cutinase.

[0044] Taking Thermobifida fusca WSH03-11 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, NaCl5g / L, pH8.0) 50 ℃, 200rpm after culturing for 20h, insert 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, cutin 1g / L, pH8.0), 50°C, 200rpm, after culturing for 50h, centrifuge the cutinase fermentation broth 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 passing through the activated carbon column for salting out overnight, centrifuge at 10000rpm for 20min at 4°C, dissolve the precipitate with an appropriate amount of buffer A (20mM Tris-HCl, pH8), add 20% ammonium sulfate, 0.22 Samples were prepared after filtration with a μm membrane. After t...

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 sequencing was ANPYERGP, which was the same as the protein Tfu_0883. The protein Tfu_0883 was predicted to be a triglyceride enzyme through sequence prediction , without indication of its cutinase activity. The pure wild fungus cutinase is subjected to enzymatic hydrolysis of cutin (apple peel extracted with chloroform and methanol, treated with 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, add glacial acetic acid to terminate the reaction after the reaction, extract the product fatty acid with chloroform, and carry out methylation and silylation, use GC -MS mea...

Embodiment 3

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

[0048] The Thermobifida fusca strain was cultured in LB liquid medium (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L) for 2 days, and the bacterial cells were collected by centrifugation at 10000rpm, washed with sterile water, and the collected precipitate was suspended in 500 μL Tris-EDTA (three (Hydroxymethylaminomethane-ethylenediaminetetraacetic acid) buffer solution, add 15 μL lysozyme, incubate at 37°C for 30 minutes, then add 5 μL RNase, incubate at 37°C for 30 minutes, add 30 μL 10% SDS (sodium dodecyl sulfate) and 15 μL of proteinase K, incubated at 37°C for 60 min, added 100 μL of NaCl (5M) and 80 μL of CTAB (cetyltrimethylammonium bromide), incubated at 65°C for 20 min, and used 700 μL of phenol: chloroform: isoamyl alcohol ( 25:24:1) extraction, 10000rpm centrifugation, supernatant was extracted with 700μL chloroform: isoamyl alcohol (24:1), 10000rpm centr...

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Abstract

The invention relates to heat resistant exogenous enzyme, and the coding gene and the expression formula thereof, and belongs to the biological engineering technical technology. The invention provides the bacteria exogenous enzyme different from the accurate exogenous enzyme, and the coding gene and the expression formula thereof. The exogenous enzyme comprises the abstraction of Thermobifida fusca WSH03-11 genomic DNA, the design of primer, and the gene of the heat resistant exogenous enzyme obtained by PCR augmenting, the exogenous enzyme is provided with SEQ ID NO: 1 showed nucleotide sequence, the total length is 906 nucleotides, the coding is 301 amino acids, so as to take plasmid pET20b (+) as the expression carrier, and to take E.coli BL21 Rosetta (DE3) PlysS as expression host, and the high-efficient expression of the heat resistant exogenous enzyme agene can be realized. The thermal stability of the exogenous enzyme is good, and the invention which has good hydrolytic action to cutin, triglyceride and various solubility synthesized greases can be widely used in industries such as spinning and cleaning agent.

Description

technical field [0001] The invention relates to yi, gif heat-resistant cutinase and its coding gene and expression, belonging to the technical field of bioengineering. Background technique [0002] Cutinases are hydrolytic enzymes capable of hydrolyzing macromolecules of cutin. As a kind of 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, cutinases have hydrolytic activity, and cutinases can perform transesterification of fats and oils or alcohols (stereo ) Selective esterification; can carry out esterification and transesterification reactions between butyric acid and 2-butanol, methyl-, ethyl-, propyl propionate and other substances, and can also participate in the synthesis of polymer surface activity ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/18C12N15/55C12N1/21C12R1/19
Inventor 陈坚吴敬陈晟
Owner JIANGNAN UNIV
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