Novel esterase, encoding gene and application thereof in splitting (+/-)-1-phenethyl alcohol and (+/-)-styralyl acetate

A technology of styroyl acetate and phenylethyl alcohol, applied in application, genetic engineering, plant gene improvement, etc., can solve the problems of low lipase activity, long reaction time, high energy consumption, etc., and achieve strong stereoselectivity and enantiomeric excess The effect of high value and mild reaction conditions

Active Publication Date: 2015-10-07
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
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Problems solved by technology

[0005] However, most of the reported methods for preparing optically pure 1-phenylethanol are to use racemic 1-phenylethanol and vinyl acetate as substrates in a non-aqueous phase system for resol...

Method used

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  • Novel esterase, encoding gene and application thereof in splitting (+/-)-1-phenethyl alcohol and (+/-)-styralyl acetate
  • Novel esterase, encoding gene and application thereof in splitting (+/-)-1-phenethyl alcohol and (+/-)-styralyl acetate
  • Novel esterase, encoding gene and application thereof in splitting (+/-)-1-phenethyl alcohol and (+/-)-styralyl acetate

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Embodiment 1

[0039] 1. Isolation and identification of Bacillus

[0040] Bacillus sp. SCSIO 15121 was isolated from the Indian Ocean (89°29.22′E, 10°00.12′N)-3400m deep sea mud, and the cultured bacteria were used for the extraction of total DNA.

[0041] 2. Cloning and expression of esterase EST01281 gene and preparation of enzyme powder

[0042] According to the analysis of the EST01281 gene sequence in the whole genome, corresponding primers were designed to amplify the gene and connected to the expression vector pET-28a(+), and then introduced into Escherichia coli BL21(DE3) for high-efficiency expression.

[0043] Design the primers as follows: the upstream primer is 5′-TGCTAGC CATATG TCAAACCATTCACCGATTACCC-3′; downstream primer is 5′-C GAATTC TTATTTACTGAAAAAACGTAATATAC-3', NdeI and EcoRI restriction sites (underlined parts) were designed at the 5' ends of the upstream and downstream primers respectively, and the genomic DNA of Bacillus sp. SCSIO 15121 was used as a template for P...

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Abstract

The invention discloses novel esterase, encoding gene and an application thereof in splitting (+/-)-1-phenethyl alcohol and (+/-)-styralyl acetate. The amino acid sequence of the esterase is shown in SEQ ID NO.2 and the nucleotide sequence is shown in SEQ ID NO.1. Asymmetric hydrolysis is carried out on the (+/-)-styralyl acetate in an aqueous phase and (S)-styralyl acetate with the enantiomer excess value being 95% and (R)-1-phenethyl alcohol with the enantiomer excess value being more than 99% are obtained; and asymmetric transesterification is carried out on the (+/-)-1-phenethyl alcohol in an organic phase and (R)-styralyl acetate with the enantiomer excess value being 99% and (S)-1-phenethyl alcohol with the enantiomer excess value being more than 70% are obtained. Compared with the traditional chemical splitting phase, the novel esterase, the encoding gene and the application disclosed by the invention have the advantages of mild reaction condition, no pollution and high enantiomer excess value and the obtained product can be used for synthesizing corresponding medicines after being simply purified.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering and biocatalysis, and specifically relates to a novel esterase and its coding gene and its application in splitting (±)-1-phenylethyl alcohol and (±)-styroyl acetate. Background technique: [0002] Esterase is a general term for enzymes that can catalyze the hydrolysis and synthesis of ester bonds. Glycerol and fatty acids are produced when catalyzing the hydrolysis of ester bonds; esters are produced when catalyzing the dehydration condensation reaction between the carboxyl group of an acid and the hydroxyl group of an alcohol. Aroma substances. Esterase widely exists in animals, plants and microorganisms, among which animal pancreas esterase and microbial esterase are the main sources of esterase. At present, esterase has been widely used in the fields of food brewing, agriculture, pharmaceutical chemistry, pulp and paper industry, sewage treatment and bioremediation. [0003] Chi...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12P41/00C12P7/62C12P7/22
CPCC12N9/18C12P7/22C12P7/62C12P41/003C12Y301/01001
Inventor 胡云峰梁甲元张云孙爱君邓盾
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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