Esterase and applications thereof

A technology of esterase and ferulic acid esterase, applied in application, enzyme, hydrolase and other directions, can solve the problems of no gene and protein sequence, report, etc.

Active Publication Date: 2016-11-30
卓虹超源生物科技(郑州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that Bacillus also has ferulic acid esterase activity (J.DonaghyáP.F.KellyáA.M.McKay(1998). "Detection of ferulic acid ester

Method used

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  • Esterase and applications thereof
  • Esterase and applications thereof
  • Esterase and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] This example is the cloning of the esterase gene of the present invention and the construction of Escherichia coli engineering bacteria.

[0022] 1. Extraction of Bacillus sp.SYBC hb4DNA

[0023] Cultivate the Bacillus sp.SYBC hb4 strain in LB medium for 12 hours, centrifuge at 12,000rmp / min for 10 minutes to obtain the bacteria, and use the bacterial genome DNA extraction kit (TaKaRa company) to extract the genome of the Bacillussp.SYBC hb4 bacteria according to its operation Total DNA, refrigerated for later use.

[0024] 2. Competent preparation of Escherichia coli

[0025] (1) Inoculate E. coli DH5α and BL21(DE3) into 250 mL shake flasks containing 20 mL LB medium respectively, and culture overnight at 37° C. and 200 rpm / min.

[0026] (2) Inoculate in 50 mL LB medium according to 1% inoculum amount, and culture at 37°C until OD600 is about 0.6 (about 2-3 hours).

[0027] (3) Transfer the bacterial solution to a 50 mL pre-cooled centrifuge tube, place on ice for 3...

Embodiment 2

[0055] This example is the induced expression and separation and purification of the esterase of the present invention.

[0056] 1. Add 500μl of recombinant bacteria solution to a 50ml shake flask. Cultivate at 37°C for 2.5h, and stand at 15°C for 0.5h. Then add 20 μl of 0.5M IPTG, and incubate at 15°C for 24 hours. Centrifuge the fermentation broth (8,000rmp / min, 10min) to obtain the bacteria, redissolve the bacteria with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (20mmol / L, pH7.0), break it with an ultrasonic breaker, and centrifuge (8,000 rpm / min, 10min) to collect the supernatant to obtain the crude enzyme solution.

[0057] 2. Use the AKTA avant 150 protein purification system to purify the crude enzyme liquid obtained in step 1 with a nickel column. The elution method is: put the four pipelines A1, A2, B1, and B2 into water, and set the system flow to 20ml / min flow rate, exhaust. Then set system flow 1ml / min, flow path (column position 3...

Embodiment 3

[0059] This example shows the optimum temperature and temperature stability of the esterase of the present invention. Using methyl ferulate as the substrate, the substrate and the phosphate buffer solution with a pH of 7.0 were placed in a water bath at 25-70°C for 1 hour to measure the enzyme activity of the esterase, and the optimum reaction temperature of the enzyme was determined to be 40°C .

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Abstract

The invention relates to obtaining, cloning expression and applications of an esterase gene, belongs to the field of biological engineering, and discloses substrate properties, wherein the esterase has wide effects and important industrial application values.

Description

technical field [0001] The invention clones and expresses an esterase, discloses its nucleotide sequence, amino acid sequence, enzymatic property and application, and belongs to the field of industrial microorganisms. Background technique [0002] Feruloyl esterases (Feruloyl esterases, FAEs), also known as cinnamic esterases, are a carboxylesterase that can hydrolyze esters in methyl ferulate, oligosaccharide esterase and polysaccharide ferulic acid ester bond, an enzyme that releases free ferulic acid. It can cut off the polysaccharide-polysaccharide and polysaccharide-lignin crosslinks in the cell wall, which is beneficial to the degradation of polysaccharides in the cell wall material and the release of lignin, so it has broad application prospects in the food, feed and paper industries. In the food industry, esterase is used to degrade the ferulic acid ester bond in the plant cell wall to obtain free ferulic acid with medicinal value and health care function. The plan...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55
CPCC12N9/18C12Y301/01073
Inventor 蔡宇杰王小梅陈佳君廖祥儒白亚军郑晓晖
Owner 卓虹超源生物科技(郑州)有限公司
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