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Ethyl chrysanthemate esterase and coding gene and specific engineering baterium for expression and uses of the enzyme

A technology of ethyl chrysanthemum acid esterase and coding gene, which is applied in the direction of genetic engineering, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of low enzyme activity of ethyl chrysanthemum acid esterase, and achieve high practicability And the effects of generalizability, easy industrialization, and broad application prospects

Inactive Publication Date: 2009-04-22
XIAHAN BIOENG SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They used the ethyl chrysanthemic acid esterase of Arthrobacter globiformis to ferment and express it in Escherichia coli, and used the produced chrysanthemic acid ethyl esterase to biosynthesize (+)-trans chrysanthemic acid, but the obtained ethyl chrysanthemic acid The enzyme activity of esterase is low (Masako nishizawa, Masatoshi shimizu, Hideo ohkawa, and Masaharu kanaoka. Steroselective production of (+)-trans-chrysanthemic acid by a microbial esterase: cloning, nucleotide sequence, and overexpression of the esterasegene of Arthrobacter globiformis in Escherichia coli. Applied And Environmental Microbiology. 1995, 61: 3208-3215.)

Method used

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  • Ethyl chrysanthemate esterase and coding gene and specific engineering baterium for expression and uses of the enzyme
  • Ethyl chrysanthemate esterase and coding gene and specific engineering baterium for expression and uses of the enzyme
  • Ethyl chrysanthemate esterase and coding gene and specific engineering baterium for expression and uses of the enzyme

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Embodiment 1

[0040] Embodiment 1, the cloning of ethyl chrysanthemum acid esterase gene ECE1

[0041] 1. PCR amplification of ethyl chrysanthemic acid esterase gene

[0042] According to the nucleotide sequence (GenBank number: L22516) of the disclosed ethyl chrysanthemic acid esterase gene, primers are designed, and the primer sequences are as follows:

[0043] ECE-UP (upstream primer): 5'-GAGCT CCATGG ATGCACAGACGATTGCC-3';

[0044] ECE-Down (downstream primer): 5'-GAAGACTCCTACTGAGCCAGTTCGG-3'

[0045] Wherein, the upstream primer introduces the restriction endonuclease NcoI recognition sequence (underlined base), and changes the start codon "GTG" of the chrysanthemum acid ethyl esterase gene to "ATG". Using the genomic DNA of Arthrobacter globiformis as a template, under the guidance of primer ECE-UP and primer ECE-Down, PCR amplified ethyl chrysanthemum acid esterase gene. The PCR reaction system is: 1 μl of Arthrobacter annulus bacteria solution treated with ultrasonic treatment f...

Embodiment 2

[0052] Embodiment 2, the construction of special-purpose expression engineering bacteria for ECE1

[0053] 1. Construction of the special expression vector pMA5-ECE1 for ECE1

[0054] Such as figure 2 As shown, the construction method of the special expression vector pMA5-ECE1 for ECE1 is as follows: Precipitate with absolute ethanol and recover the ECE1 amplified by PCR in Example 1, cut it with restriction endonucleases NdeI and BglII, and combine with T 4 DNA ligase ligation, the ligation product was transformed into Escherichia coli DH5α, and the transformant was selected to extract the plasmid after screening, and the restriction endonuclease NdeI and XbaI were used for enzyme digestion and identification, and the 1100bp fragment obtained by enzyme digestion was a positive clone, and then positive The cloned plasmid was further identified by PCR. The primers used were primer 1 and primer 2 in Example 1, and the PCR product was sequenced. The sequencing results showed t...

Embodiment 3

[0057] Embodiment 3, ECE1 expression in Bacillus subtilis and the mensuration of enzyme activity

[0058] 1. Expression of ECE1 in Bacillus subtilis

[0059]Common Bacillus subtilis medium formula: 2-3% yeast extract, 1-2% peptone, 0.1-0.2% K per 1000mL water 2 HPO 4 .3H 2 O, 0.1% KH 2 PO 4 , 1% NaCl.

[0060] Inoculate the BsMA5-ECE1 special-purpose expression engineered bacterium BsMA5-ECE1 constructed in Example 2 in common Bacillus subtilis culture medium, and continuously culture it at 37°C for 120 hours to transform Bacillus subtilis DB1342 with pMA5 empty vector and Escherichia coli transformed with ECE1 BL21(DE3)plys (cloning ECE1 into the vector pET26 (Navagen Company) to obtain an Escherichia coli expression vector containing ECE1, named pET26-ECE1, and then transforming pET26-ECE1 into Escherichia coli BL21(DE3)plys, obtained by screening Positive clone transformant, the fermentation culture condition is 12h under 37 ℃.) as the control. On the third day, samp...

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Abstract

The invention discloses a chrysanthemic acid carbethoxyl esterase with its coding gene and special expression engineering fungus, which comprises the protein with one of following amino acid residue sequence: 1) SEQ ID No. 1 and 2 in sequence table; 2) substituting, deleting or adding to the residue of SEQ ID No. 1 by one to ten amino acid residues as well as with the activity of chrysanthemic acid carbethoxyl esterase. The modified gene can express chrysanthemic acid carbethoxyl esterase in bacillus subtilis with highlevel and constructs opposite expression engineering fungus. The experiments show: this esterase can undivided act on and generate (+)-trans- chrysanthemic acid ethyl ester. This invention has strong practice and wide application.

Description

technical field [0001] The present invention relates to an enzyme and its encoding gene and its expression special engineering bacteria and the application of the enzyme, in particular to a kind of chrysanthemum acid ethyl esterase and its encoding gene and its expression special engineering bacteria and the enzyme in the production of (+)-trans Application of chrysanthemic acid. Background technique [0002] Chrysanthemic acid (Chrysanthemic acid, [2,2-dimethyl-3-(2-methyl-1-propenyl) cyclopropane carboxylic acid], molecular structure is shown in formula 1) is a new type, high efficiency, low toxicity An important intermediate of pyrethroid insecticides, it is widely used in the synthesis of such insecticides because it is the acidic part of synthetic pyrethroid insecticides. In addition, chrysanthemic acid is also one of the most popular whitening ingredients in the world, which can effectively inhibit the activity of tyrosinase. [0003] [0004] ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/56C12N1/21C12P7/40C12R1/125
Inventor 夏国兴邱并生胡学博
Owner XIAHAN BIOENG SHANGHAI
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