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Lipase mutant deriving from talaromyces thermophilus, coding gene and application thereof

A technology for encoding genes and mutants, applied in the application field of 2-carboxyethyl-3-cyano-5-methylhexanoic acid, can solve the problem of restricting large-scale industrial applications, harsh production conditions, and the amount of organic solvents used Large and other problems, to achieve the effect of good application prospects

Active Publication Date: 2017-05-17
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical synthesis of pregabalin has disadvantages such as low atom economy, large amount of organic solvents, and harsh production conditions, which limit its large-scale industrial application

Method used

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  • Lipase mutant deriving from talaromyces thermophilus, coding gene and application thereof
  • Lipase mutant deriving from talaromyces thermophilus, coding gene and application thereof
  • Lipase mutant deriving from talaromyces thermophilus, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of Lipase Mutation Library

[0031] The pET-28b-TTL plasmid, which was cloned from the lipase gene of Talaromyces thermophilus (GenBank accession No.JF414585.1) and linked to pET-28b(+) as a template (nucleotide sequence SEQ ID NO.2 ), using T7promoter and T7terminator as primers (Table 1) for PCR amplification, and randomly introducing mutations. PCR reaction system (50μL): template 0.5~20ng, 1×Taq Buffer (without Mg 2+ ), 0.2mM dGTP, 0.2mM dATP, 0.1mM dCTP, 0.1mM dTTP, 0.2mMMnCl 2 , Primers T7promoter and T7terminator each 0.2μM, Taq DNA polymerase 5U. PCR conditions (1) 95°C pre-denaturation for 3 minutes; (2) 95°C denaturation for 30s; (3) 55°C annealing for 30s; (4) 72°C extension for 1 minute, steps (2) to (4) total 30 cycles; (5) ) Finally, extend at 72°C for 10 minutes and store at 4°C. After the PCR product was analyzed by agarose gel electrophoresis, the gel was cut and recovered, and it was digested with NcoI and Xhol, ligated with the s...

Embodiment 2

[0034] Example 2 High-throughput screening of mutants

[0035] Pick the single colony in Example 1 and culture it in a 96 deep-well plate, add 200μL LB medium (containing 50μg / mL kanamycin) to each well plate, and incubate at 37°C for 2.5h to OD 600 When it is about 0.6, add the final concentration of 0.1mM IPTG to induce, and induce culture at 28℃ for 20h. Centrifuge the bacteria in the 96-deep well plate for 15 min (5000 rpm, 4°C), discard the supernatant, and resuspend the bacteria in Tris-HCl (20 mM, pH 8.0) buffer as the biocatalyst for the reaction. The bacterial suspension was added to each well of a 96 shallow-well plate, 200 μL per well, then the substrate CNDE (final concentration 100 mM) was added, and 0.01% bromothymol blue was used as an indicator, and reacted at 30°C for 1-3 hours.

[0036] According to the speed of the color change, the bacteria with increased vitality were selected (using the wild-type strain as the control), and the catalytic activity of CNDE was v...

Embodiment 3

[0037] Example 3 Site-directed mutation and screening

[0038] In order to further improve the enzyme activity of the mutants, site-directed mutagenesis is used to continue to screen for better complex mutants. Using the expression plasmid pET-28b-L259F as a template, site-directed mutagenesis was performed through whole plasmid amplification. The PCR system (50μL) is: template 0.5-20ng, 5×PrimeSTAR Buffer 10μL, dNTP Mixture 4μL, mutation primer (Table 1) each 1μL, PrimeSTAR DNA Polymerase 0.5μL. PCR conditions (1) 95°C pre-denaturation for 3 minutes; (2) 95°C denaturation for 15 seconds; (3) 55°C annealing for 10 seconds; (4) 72°C extension for 5 minutes, steps (2) to (4) total 30 cycles; (5) ) Finally, extend at 72°C for 10 minutes and store at 4°C. The PCR product was verified by agarose gel electrophoresis, digested with DpnI, and then introduced into E.coli BL21(DE3), and spread on an LB plate containing 50μg / mL kanamycin. The catalytic activity of each mutant on CNDE was...

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Abstract

The invention discloses a lipase mutant deriving from talaromyces thermophilus, a coding gene and application thereof in hydrolysis of racemic 2-carboxyethyl-3-cyano-5-methyl ethyl hexanoate to synthesize (3S)-2-carboxyethyl-3-cyano-5-methyl hexanoate. The mutant is obtained by mutation of one or more 206, 207 and 259 amino acids of an amino acid sequence shown in SEQ ID No.1. By means of protein molecule modification, the activity of TTL stereoselectivity CNDE hydrolysis is improved from 4.50U / mg to 160.55U / mg, and is much higher than the activity of lipase CNDE hydrolysis reported currently. Therefore, the mutant obtained in the invention has a great application prospect in high-efficient CNDE catalysis to synthesize pregabalin chiral intermediate (3S)-2-carboxyethyl-3-cyano-5-methyl hexanoate.

Description

[0001] (1) Technical field [0002] The invention relates to a lipase and its application, in particular to a lipase mutant derived from Talaromyces thermophilus, coding gene and asymmetric resolution of 2-carboxyethyl-3-cyano-5-methyl The application of ethyl caproate in the synthesis of pregabalin, the key chiral intermediate (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid. [0003] (2) Background technology [0004] Pregabalin (Pregabalin, PGB), trade name The chemical name is (S)-(+)-3-aminomethyl-5-methylhexanoic acid, which is a calcium ion channel modulator, which can effectively inhibit neuropathic pain, epilepsy and other related neuronal hyperexcitement and Diseases with increased neurotransmitter release (Curr. Opin. Pharmacol. 2006, 6: 108-113). It has been approved by the FDA for the treatment of post-herpetic neuralgia, diabetic peripheral neuralgia, epilepsy, fibromyalgia and other diseases. Compared with traditional similar drugs such as gabapentin, pregabalin is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12P41/00C12P13/00
CPCC12N9/20C12P13/002C12P41/005C12Y301/01003
Inventor 郑仁朝郑裕国丁旭汤晓玲
Owner ZHEJIANG UNIV OF TECH
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