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Esterase and application thereof

A technology of esterase and ferulic acid esterase, applied in the fields of application, enzyme, hydrolase, etc., can solve the problems of unknown gene and protein sequence, report, etc.

Active Publication Date: 2017-02-22
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 esterase production by Bacillus spp.and lactobacilli."Appl Microbiol Biotechnol 50:257-260 ), but there is no related gene and protein sequence report

Method used

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  • Esterase and application thereof
  • Esterase and application thereof
  • Esterase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

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

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

[0023] The Bacillus sp.SYBC hb4 strain was cultured in LB medium for 12 hours and centrifuged at 12,000rmp / min for 10 minutes to obtain the bacteria. Bacterial genomic DNA extraction kit (TaKaRa) was used to extract the Bacillus sp.SYBC hb4 bacterial genome according to its operation. Total DNA, put in the refrigerator for later use.

[0024] 2. Preparation of Escherichia coli Competent

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

[0026] (2) Inoculate 1% of the inoculum into 50mL LB medium, culture at 37°C to an OD600 of about 0.6 (about 2-3h).

[0027] (3) Transfer the bacterial solution to a 50mL pre-cooled centrifuge tube, place it on ice for 30min, centrifuge at 8000rpm / min, 4℃ for 5min.

...

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 bacterial solution to a 50ml shake flask. Incubate at 37°C for 2.5h, and stand at 15°C for 0.5h. Then add 20μl 0.5M IPTG, cold induce culture at 15℃ for 24h. The fermentation broth was centrifuged (8,000rmp / min, 10min) to obtain the bacteria, and the bacteria were reconstituted with disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (20mmol / L, pH 7.0), broken by an ultrasonic disruptor, and centrifuged (8,000 rpm). rmp / min, 10min) collect the supernatant to obtain the crude enzyme solution.

[0057] 2. Use the AKTA avant 150 protein purification system to operate the crude enzyme solution obtained in step 1 for nickel column purification. The elution method is: put all four pipes A1, A2, B1, and B2 into the water and set the system flow 20ml / min flow rate, exhaust. Then set system flow 1ml / min, flow pa...

Embodiment 3

[0059] This example shows the optimum temperature and temperature stability of the esterase of the present invention. Using methyl ferulic acid as a substrate, the substrate and a phosphate buffer with a pH of 7.0 are subjected to a water bath at 25-70°C for 1 hour to determine the enzyme activity of the esterase, and the optimal reaction temperature of the enzyme is determined to be 45°C .

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PUM

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Abstract

The invention relates to acquisition of an esterase gene, and cloning and expression and application thereof, which belong to the field of bioengineering. The invention discloses a substrate characteristic of the esterase gene. An esterase has a wide function and an important industrial application value.

Description

Technical field [0001] The invention clones and expresses an esterase, discloses its nucleotide sequence, amino acid sequence, enzymatic properties and applications, and belongs to the field of industrial microorganisms. Background technique [0002] Feruloyl esterases (Feruloyl esterases, FAEs), also known as cinnamic acid esterases, are a kind of carboxylesterase, which can hydrolyze the esters in methyl ferulate, oligosaccharide esterase and polysaccharide ferulate Bond to release free ferulic acid. It can cut off the cross-links between polysaccharide-polysaccharide and polysaccharide-lignin in the cell wall, which is beneficial to the degradation of polysaccharide in the cell wall material and the release of lignin. Therefore, it has broad application prospects in the food, feed and paper industries. In the food industry, the use of esterase to degrade the ferulic acid ester bond in the plant cell wall can obtain free ferulic acid with medicinal value and health care functi...

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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