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Esterase as well as encoding genes and application thereof

A technology of esterase and gene, which is applied in the field of esterase and its coding gene and application, and can solve problems such as the inability to obtain S-type products

Active Publication Date: 2015-04-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzymatic resolution of the above-mentioned routes cannot obtain the S-type product with higher optical purity (ee>99%)

Method used

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  • Esterase as well as encoding genes and application thereof
  • Esterase as well as encoding genes and application thereof
  • Esterase as well as encoding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The construction of the recombinant plasmid of embodiment 1 esterase gene

[0054] (1) Cloning of esterase gene

[0055] According to the genome DNA sequence of Pseudomonas (Pseudomonas) CGMCC NO.4184, a pair of primers were designed, and the primer sequences were as follows:

[0056] The upstream primer is: 5'-GGGAATTC CATATG CAGATCCAGGGTCACTATGAGCTGAAGTTCG-3', (as shown in SEQ ID NO.3), wherein the underlined part is the Nde I restriction site;

[0057] The downstream primer is: 5'-CCC CTCGAG AAGGCAAGTGCCAAGAACGCGTACCA-3', (as shown in SEQ ID NO.4), wherein the underlined part is the Xho I restriction site.

[0058] Using Pseudomonas sp. (Pseudomonas sp.) CGMCC NO.4184 genomic DNA as a template for PCR amplification, the PCR reaction system and reaction conditions are as follows:

[0059] PCR amplification system:

[0060]

[0061] PCR amplification conditions:

[0062] 1) Pre-denaturation: 95°C for 5 minutes;

[0063] 2) Denaturation: 98°C for 10s; Anneal...

Embodiment 2

[0072] Example 2 Construction of recombinant E.coli BL21(DE3) to induce expression of esterase

[0073] The recombinant cells carrying the pET30a-estZF172 plasmid were inoculated in 5 mL LB liquid medium (containing 50 g / mL kanamycin), and cultured overnight at 37° C. and 200 rpm. The above culture was inoculated in 50mL LB liquid medium (containing 50g / mL kanamycin) at a ratio of 1%, and cultivated to OD at 37°C and 200rpm. 600 When reaching 0.6-0.8, add IPTG to a final concentration of 0.5mmol / L. The culture was induced at 18°C ​​and 200 rpm. After 12 hours, the cells were collected by centrifugation at 12,000×g, washed twice with pre-cooled Tris-HCl buffer (300 mM, pH 8.5), and the obtained wet cells were stored at 4°C.

Embodiment 3

[0075] Resuspend the corresponding wet cells with 14mL Tris-HCl buffer (300mM, pH 8.5) at a ratio of 0.085‰ (0.085g dry cells / L), divide into 7 parts, each 2mL, add the substrate rac-2-carboxylate Ethyl-3-cyano-5-methylhexanoate such that ethyl rac-2-carboxyethyl-3-cyano-5-methylhexanoate relative to rac-2-carboxyethyl-3 -The mass concentration of ethyl cyano-5-methylhexanoate and the total amount of cell suspension was 5g / L. Put the 7 parts of the solution into a constant temperature shaker with a temperature of 20°C, 30°C, 35°C, 40°C, 45°C, 50°C and 55°C, respectively, at a speed of 200 rpm. After 1 hour, the gas phase detection method was used to detect the substrate and The concentration of the product, its retention time on the gas phase as Figure 5 As shown, peak 1 is 2-carboxyethyl-(R)-3-cyano-5-methylhexanoic acid decarboxylation (11.46min); peak 2 is 2-carboxyethyl-(S)-3-cyano Base-5-methylhexanoic acid decarboxylate (12.53min); peak 3 is 2-carboxyethyl-(R)-3-cyano...

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Abstract

The invention discloses esterase as well as encoding genes and application thereof. The amino acid sequence of the esterase is shown in SEQ ID NO.2. The esterase genes are obtained by cloning from pseudomonas Pseudomonas CGMCC NO.4184, and the esterase expressed by the genes has the characteristics of high expression quantity, high selectivity and chiral selectivity. The engineering bacteria containing the esterase genes are applied to a method for catalyzing hydrolysis of rac-2-carboxyethyl-3-cyano-5-methyl ethyl caproate so as to prepare 2-carboxyethyl-(S)-3-cyano-5-methyl ethyl caproate, and when the conversion rate of the reaction is controlled to exceed 50 percent, the unhydrolyzed high-purity 2-carboxyethyl-(S)-3-cyano-5-methyl ethyl caproate can be obtained.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an esterase, its coding gene and application. Background technique [0002] 2-Carboxyethyl-(S)-3-cyano-5-methylhexanoic acid ethyl ester is chemically enzymatically synthesized Pregabalin (PGB for short, trade name ) is an important chiral intermediate. Pregabalin is a three-position isobutyl substitution of the neurotransmitter γ-aminobutyric acid (GABA). As a new generation of antiepileptic drugs, it has been used to treat a variety of central nervous system disorders, including neuropathic Pain, Social Anxiety Disorder, Generalized Anxiety Disorder and adjuvant treatment of focal partial seizures, etc. Due to the obvious advantages of this drug compared with other similar drugs for the treatment of epilepsy, the drug produced and marketed by Pfizer The annual sales are rising year by year, reaching US$3.69 billion, US$4.16 billion and US$4.60 billion in 2011, 2012 and 2...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21C12P13/00
CPCC12N9/18C12N15/70C12P13/00
Inventor 杨立荣许方馨吴坚平徐刚
Owner ZHEJIANG UNIV
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