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Thermostable carboxylesterase gene, coding protein and application thereof

A carboxylesterase, thermostable technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of less active protein, lack of post-translational modification system, etc.

Inactive Publication Date: 2009-12-16
ANHUI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Especially Escherichia coli, as a lower prokaryotic organism, lacks a complete post-translational modification system, and the expression of esterase in it often exists in the form of inclusion bodies, and rarely obtains active protein. Thermostable esterases from mesophilic strains in hot or hyperthermophilic environments have not been reported

Method used

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  • Thermostable carboxylesterase gene, coding protein and application thereof
  • Thermostable carboxylesterase gene, coding protein and application thereof
  • Thermostable carboxylesterase gene, coding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1. Acquisition of carboxylesterase gene estW

[0023] Cultivate Streptomyces lividans TK64 and extract the genome, use its genomic DNA as a template, and use estW-F: ATACGTC Catatgag TTTCCTCAATCCCCGCAT and estW-R:TAA CTCGAG CGATTATAAAGCTTCCCG (the underlines are NdeI and XhoI restriction sites respectively) as primers, PCR amplification was carried out under the conditions of 98°C, 10s, 55°C, 10s, 72°C, 1min 10s, 35 cycles, and the products were agarose gel Electrophoresis ( figure 1 ), subsequently, the amplified product and the vector pET28b(+) were digested by NdeI and XhoI respectively, then ligated, transformed into E. cut identification ( figure 2 ); the expression plasmid pET28b-estW is sequenced, and the amino acid sequence is shown in SEQ ID NO:1.

[0024] 2. Construction of expression engineering bacteria

[0025] The expression plasmid pET28b-estW was transformed into Escherichia coli Rosetta (DE3) competent cells according to the existing technology,...

Embodiment 2

[0031] EstW optimum temperature detection:

[0032] EstW enzyme activity detection standard system 1ml, 50mM Tris-HCL (pH 8.0), 1% acetonitrile, 0.5mM p-nitrophenol hexanoate, 5μl pure enzyme solution, using a Cary 300 UV spectrophotometer with automatic temperature control , the detection wavelength is 410nm. The reaction temperature (10°C-60°C) was set, the test was repeated 3 times independently, and the average value was taken. The results showed that the optimum temperature of the enzyme was 50°C ( Figure 4 ).

Embodiment 3

[0034] EstW optimal pH detection

[0035]Using a standard assay system, the reaction temperature is 50°C, and the corresponding buffer solution is used in the corresponding pH range, pH5.0 to 7.04, 50mM sodium citrate buffer; pH 7.0-9.0, 50mM Tris·HCl buffer; pH 8.5-9.0, 50mM K 2 HPO 4 -KOH buffer. Detection wavelength is OD 348 , the experiment was repeated three times independently, and the results showed that the optimum pH of the enzyme was 8.0 ( Figure 5 ).

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Abstract

The invention discloses a thermostable carboxylesterase gene, coding protein and application thereof, the esterase has amino acid sequence shown in SEQ ID NO:1. Compared with the prior art, the esterase containing EstW is thermostable esterase obtained from streptomycete in known mesophilic bacteria, can be widely applied to production of non-steroidal anti-inflammatory drugs or ferulic acid.

Description

technical field [0001] The invention relates to a carboxylesterase gene, encoded protein and application thereof, in particular to a thermostable carboxylesterase gene, encoded protein and application thereof. Background technique [0002] Esterases (EC 3.1.1.X) are a large class of enzymes that can catalyze the hydrolysis and formation of ester bonds, and are widely distributed in animals, plants and microorganisms. Esterases mainly include carboxylesterases, carboxylester hydrolases, EC 3.1.1.1) and lipases (lipase, triacylglycerol hydrolases, EC3.1.1.3). Carboxylesterase can hydrolyze or synthesize glycerolipids with acyl chain length less than 10, using tributyryglycerol as the standard substrate, while lipase generally uses glyceride with acyl chain length greater than or equal to 10 carbon atoms as Substrate, triolein is its standard substrate, but most lipases can also hydrolyze the substrate of carboxylesterase. Esterases belong to the typical α / β hydrolase superfa...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12P7/42C12P1/00C12R1/465
Inventor 朱国萍王宝娟王鹏葛亚东曹正宇苏蕊蕊陈露露宋平
Owner ANHUI NORMAL UNIV
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