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High-temperature cutinase and gene order thereof

A technology of cutinase and high temperature, applied in the direction of genetic engineering, plant gene improvement, hydrolytic enzymes, etc., can solve the problems of obtaining a large number of products, the inability to use gene recombination technology, and insufficient attention to the research of bacterial cutinase, achieving high stability, The effect of efficient expression

Active Publication Date: 2010-04-14
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Based on the research at home and abroad, it has been found that foreign research on cutinases mainly focuses on fungal cutinases, while there are few reports on bacterial cutinases. The main reason is that foreign countries still focus on the development of fungi whose biochemical properties have been deeply studied Cutinase, the study of bacterial cutinase has not been paid enough attention; the gene encoding bacterial cutinase has not been elucidated, and it is impossible to use gene recombination technology to obtain a large number of products as research materials

Method used

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  • High-temperature cutinase and gene order thereof
  • High-temperature cutinase and gene order thereof
  • High-temperature cutinase and gene order thereof

Examples

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Effect test

Embodiment 1

[0041] This example illustrates the extraction of total DNA from Thermobifida fusca WSH03-11.

[0042] The Thermobifida fusca WSH03-11 strain was cultured in LB liquid medium (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L) for 2 days, collected by centrifugation at 10000rpm, washed with sterile water, and the collected precipitate was suspended in 500μL Tris -EDTA (trishydroxymethylaminomethane-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 (dodecane Sodium sulfate) and 15 μL proteinase K, incubated at 37°C for 60 min, added 100 μL NaCl (5M) and 80 μL CTAB (cetyltrimethylammonium bromide), incubated at 65°C for 20 min, and incubated with 700 μL of phenol:chloroform: Extract with a mixed solvent of isoamyl alcohol at a volume ratio of 25:24:1, centrifuge at 10,000 rpm, extract the supernatant with 700 μL of chloroform:isoamyl alcohol at a volume ratio ...

Embodiment 2

[0044] This example illustrates the cloning procedure for the gene encoding a high-temperature cutinase.

[0045] Thermobifida fusca WSH03-11 total DNA was used as a template, the following nucleotide sequences were used as primers, and the underlined restriction sites Nco I and EcoR I were used to amplify the high-temperature cutinase gene by PCR.

[0046] Primer 1: 5'-ggAATACCATATgT CCATgg CCAACCCCTACgAgCgCgg-3'

[0047] Primer 2: 5'CATCTCgAgA wxya gggAACgggCAggTggAgCg-3'

[0048] The PCR reaction was carried out in a 50 μL system. The reaction conditions were 35 cycles of denaturation at 95°C for 5 minutes, denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 1 minute. A 780bp PCR fragment was amplified and recovered by tapping the rubber. The recovered fragment was ligated with the pMD18-Tsimple vector, the ligated product was transformed into Escherichia coli JM109, and the transformed product was coated on an LB plate containi...

Embodiment 3

[0050] This example illustrates the modification procedure of the high temperature cutinase gene.

[0051] Since there is an Nco I restriction site inside the target gene, and the two ends of the gene are respectively Nco I and EcoR I sites, primers were designed to remove the mutation of the Nco I site to facilitate the cloning and expression in the next step. Use the CUT-pMD18-T simple linked to the cutinase gene as a template for site-directed mutagenesis PCR, and design primers:

[0052] Primer 3: 5'-CATgggCCACTCAATgggCggCggCggC-3'

[0053] Primer 4: 5'-gCCgCCgCCgCCCATTgAgTggCCCATg-3'

[0054] The PCR reaction was carried out in a 50 μL system. The reaction conditions were 35 cycles of denaturation at 95°C for 5 minutes, denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 4 minutes. The PCR product was transformed into E.coli JM109 competent cells. The transformed bacteria were coated with 100mg / L ampicillin LB plate, and a singl...

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Abstract

The invention relates to high temperature cutinase and a gene sequence thereof, which belongs to the fields of enzyme gene engineering and enzyme engineering. The invention obtains a high temperaturecutinase gene SEQ ID NO:1 from thermobifida fusca WSH03-11 total DNA, the cutinase gene takes a plasmid pET 20b (+) as an expression vector and takes E. coli BL21 Rosetta (DE3)PlysS as an expression host to realize high level expression of the high temperature cutinase gene, the whole length of the cutinase gene is 783 nucleotides, the code is 261 amino acids, a prokaryotic expression plasmid is constructed, and escherichia coli expression cutinase is transformed. Recombinase has the activity of the cutinase. The most suitable temperature for the high temperature cutinase is 60DEG C, the mostsuitable pH value is 8, the high temperature cutinase has vey high thermal stability under the temperature of 60 DEG C, and the half-life is 45 hours. The high temperature cutinase is very suitable for the demands of being applied in the textile industry.

Description

technical field [0001] A high-temperature cutinase and its gene sequence, the invention belongs to the fields of enzyme genetic engineering and enzyme engineering. Specifically, it relates to a DNA sequence encoding thermobifida fusca high-temperature cutinase and its expression. Background technique [0002] Cutinase is a multifunctional enzyme that can hydrolyze cutin polymer molecules and ester bonds of various synthetic polyesters to degrade them into monomers and small molecular oligomers, and can also hydrolyze various insoluble triglycerides and water-soluble Sexual esters. The wide range of substrates of cutinase makes it have good application prospects in food, chemical industry, textile and other fields. [0003] The textile industry is a traditional industry and a pillar industry in China, and it occupies an important proportion in the gross domestic product and the total export value. However, my country's textile industry generally has problems such as low in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/31C12N15/55C12N15/70
Inventor 陈坚吴敬陈晟
Owner JIANGNAN UNIV
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