Esterases and coding gene and application thereof

A technology of esterase and gene, which is applied in the field of esterase and its coding gene and application, can solve the problems of not being environmentally friendly, relying on toxic substances, special reaction conditions, etc., and achieve high enzyme activity, high tolerance, and huge application potential Effect

Active Publication Date: 2020-05-01
山东阳成协盈生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are many shortcomings in the preparation of the required reactants by chemical synthesis. The preparation process is relatively complicated, the reaction conditions are special, and it depends on toxic substances and is not environmentally friendly. It will waste resources and increase the cost; The production is convenient and environmentally friendly, overcomes many shortcomings in the chemical synthesis steps, and has certain pertinence for the generation of products

Method used

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  • Esterases and coding gene and application thereof
  • Esterases and coding gene and application thereof
  • Esterases and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Acquisition and sequence analysis of the gene sequence encoding esterase E31-4922 derived from Antarctic soil

[0064] The source of the strain: Escherichia coli EPI300 clone E31-4922 in the metagenomic library of soil samples at site E2 near the Great Wall Station in Antarctica.

[0065] Specific steps are as follows:

[0066] 1.1 Construction of subcloning library

[0067] Use the BAC / PAC DNA extraction kit from OMEGA Company to extract the large fragment plasmid fosmid in E. coli EPI300 clone E31-4922 according to its instructions. Then the fosmid extracted was partially digested with restriction endonuclease Sau3AI (purchased from Fermentas Company) to obtain a DNA fragment of 2,000-5,000bp, which was connected to the pUC19 plasmid (purchased from BamHI) digested and dephosphorylated by BamHI. from NEB Corporation). Electroporate E.coli Top10 competent cells with the ligation reaction solution, coat LB solid plate containing 100 μg / ml ampicillin and 1%...

Embodiment 2

[0072] Example 2: Cloning, heterologous expression and separation and purification of esterase

[0073] 2.1 Using PCR to amplify the gene sequence

[0074] (1) Design two specific primers according to the gene E31-4922 sequence:

[0075] 4922F: aagaaggaga tatacatatg agctacccgg ctatcggtta ctg (SEQ ID NO. 3);

[0076] 4922R: tcgagtgcgg ccgcaagctt caggtgagaa cggctttccg agc (SEQ ID NO. 4);

[0077] Primers were synthesized by Jinan Boshang Biotechnology Co., Ltd.

[0078] (2) Using 4922F and 4922R as primers, using the fosmid where the gene E31-4922 is located as a template, amplify the target gene fragment with FastPfuDNA polymerase (purchased from Transgen);

[0079] The PCR reaction conditions were: pre-denaturation at 95°C for 2 min; then denaturation at 95°C for 20 sec, annealing at 50°C for 20 sec, extension at 72°C for 1 min, and after 30 cycles; extension at 72°C for 10 min.

[0080] (3) 1 wt% agarose gel electrophoresis was performed on the PCR amplification product, ...

Embodiment 3

[0103] Embodiment 3: the property determination of Antarctic esterase E31-4922

[0104] 3.1 Substrate specificity analysis

[0105] pNP ester substrates C2, C4, C8, C10, C12, and C14 (purchased from Sigma) with different carbon chain lengths were prepared with isopropanol.

[0106] The standard response is:

[0107] 20μl of 10mM pNPC4 substrate and 960μl of 50mM Tris-HCl (pH 8.0) mixture was preheated at 40°C for 3 minutes, then 20μl of the diluted enzyme solution was added and reacted at 40°C for 10min, and 100μl of 20wt% SDS (dodecyl Sodium sulfate) to stop the reaction, measure the OD405 value. The reaction without enzyme solution was used as blank control. The standard curve was drawn with different concentrations of pNP (purchased from Sigma).

[0108] Enzyme activity is defined as the amount of enzyme required to catalyze the hydrolysis of pNP ester substrate to produce 1 μM pNP per minute at a certain temperature, which is one enzyme activity unit (U). The results ...

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Abstract

The invention provides esterases and a coding gene and an application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. A gene for coding the esterases E31-4922 is obtained through screening and cloning from Antarctic soil; through researching expression proteins of the esterases, the inventor finds that the esterases show high degradation activity on short-chain esters, the optimum pH is 7.5, and the esterases stably exist in the pH range being 7.0-8.0; the optimum enzyme activity temperature is 80 DEG C, and the esterases maintain vitality being overthan 80% in the range of 60-90 DEG C; the esterases are thermally-stable esterases, and show higher tolerance for high temperature environment being over than 60 DEG C; and the esterases have important significance in enriching the family members of the esterases particularly the family members of high temperature resistant esterases.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to an esterase, its coding gene and application. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Esterases, also known as carboxylesterases, are enzymes that can catalyze the hydrolysis of ester bonds and the synthesis of ester bonds. When it hydrolyzes the ester bond, the ester bond will be broken, and the products are acids and alcohols; when the ester bond is synthesized, the carboxyl group of the acid and the hydroxyl group of the alcohol will be dehydrated and condensed, and the products are esters and other fragrance substances....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21C12P7/64C12P7/62C12R1/19
CPCC12N9/18C12N15/70C12P7/62C12P7/64C12Y301/01001
Inventor 周明扬刘健敏邢澍刘文杰刘晓雨武涛吴汉夔
Owner 山东阳成协盈生物技术有限责任公司
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