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Lipase mutant derived from Talaromyces thermophilic and its application

A lipase and mutant technology, which is applied to a lipase mutant derived from Talaromyces thermophilus and its application field to achieve the effect of improving the activity of the parent

Active Publication Date: 2017-04-12
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But currently only from Novozymes That is, the commercial Thermomyces lanuginosus lipase can meet the needs of industrial production

Method used

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  • Lipase mutant derived from Talaromyces thermophilic and its application
  • Lipase mutant derived from Talaromyces thermophilic and its application
  • Lipase mutant derived from Talaromyces thermophilic and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Amplification of parental genes and construction of expression vectors

[0024]The lipase gene was obtained from Talaromyces thermophilus ATCC 20186 (purchased from American Type Culture Collection, American Type Culture Collection) by RT-PCR. According to the Talaromyce thermophilus CTM10.103 lipase gene sequence (GenBank accession no. JF414585) reported in the literature, primers TTL-F and TTL-R (see Table 1) were designed, and the lipase gene was isolated from T.thermophilus ATCC20186 by RT-PCR . Total RNA of T.thermophilus ATCC 20186 was extracted by TRIzol method, using GoScript from Promega TM The reverse transcription kit is used to prepare the first strand of cDNA, and the reaction system and conditions refer to the instructions of the kit. Using the first strand of cDNA as a template, under the action of primers TTL-F and TTL-R, the lipase cDNA sequence was amplified by PCR. The amount of each component in the PCR reaction system (total volume 100...

Embodiment 2

[0025] Example 2: Site-directed saturation mutation of lipase position 83

[0026] Description of site-directed saturation mutagenesis technology reference (Current Protocols in Protein Science26.6.1-26.6.10, 2011; Appl.Microbiol.Biotechnol.2014,98:2473-2483), high-throughput screening technology reference for positive mutants (Appl. Microbiol.Biotechnol.2014,98:2473-2483). The specific process is as follows:

[0027] In order to saturate the Ser at the 83rd position in the parental amino acid sequence, design mutation primers S83-F and S83-R (see Table 1), use the plasmid pET28-TTL constructed in Example 1 as a template, and carry out full-plasmid PCR . The PCR amplification system is: 5×PS buffer 10 μL, dNTP (2.5mM each) 4 μL, mutant primers S83-F and S83-R 0.5 μL each, plasmid pET28-TTL 0.5 μL, PrimeSTAR DNA polymerase 0.5 μL, water up to 50 μL . PCR conditions were pre-denaturation at 98°C for 2 minutes, 25 cycles: 98°C for 10s, 65°C for 10s, 72°C for 6min, and finally...

Embodiment 3

[0028] Example 3: Site-directed saturation mutation of lipase mutant S83T site 58

[0029] Description of site-directed saturation mutagenesis technology reference (Current Protocols in Protein Science26.6.1-26.6.10, 2011; Appl.Microbiol.Biotechnol.2014,98:2473-2483), high-throughput screening technology reference for positive mutants (Appl. Microbiol.Biotechnol.2014,98:2473-2483). The specific process is as follows:

[0030] On the basis of the lipase mutant S83T, the Ser at position 58 was saturatingly mutated, and the mutant primers S58-F and S58-R (see Table 1) were designed, and the plasmid pET28-S83T was used as a template (see Example 2). Whole plasmid amplification. The PCR system is: 10 μL of 5×PS buffer, 4 μL of dNTP (2.5 mM each), 0.5 μL of mutant primers S58-F and S58-R, 0.5 μL of plasmid pET28-S83T, 0.5 μL of PrimeSTAR DNA polymerase, and water to 50 μL. PCR conditions were pre-denaturation at 98°C for 2 minutes, 25 cycles: 98°C for 10s, 65°C for 10s, 72°C for ...

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Abstract

The invention discloses a talaromyces thermophilus derived lipase mutant with the amino acid sequence shown in SEQ ID No.2, and the lipase mutant is obtained by mutation of serine at the 83th-site of the amino acid sequence shown in SEQ ID No.2 into threonine, or mutation of serine at the 58th-site of the amino acid sequence shown in SEQ ID No.2 into methionine while mutation of serine at the 83th-site of the amino acid sequence shown in SEQ ID No.2 into threonine; the activity of the talaromyces thermophilus derived lipase mutant is 2-5 times of the parent activity, and the talaromyces thermophilus derived lipase mutant can be used for industrial production of (3S)-2-carboxyethyl-3-cyano-5-methyl hexanoic acid for further synthesis of pregabalin.

Description

[0001] (1) Technical field [0002] The present invention relates to the cloning of lipase coding gene and the method for preparing lipase mutant by gene mutation technology, the obtained mutant and its chiral intermediate (3S)-2-carboxyethyl-3-cyanide in pregabalin Application in the preparation of base-5-methylhexanoic acid. [0003] (2) Background technology [0004] Lipase (lipase, EC 3.1.1.3), the system name is triacylglycerol acylhydrolase (triacylglycerol acylhydrolase), is a class that can not only catalyze ester hydrolysis, but also catalyze ester synthesis, transesterification, aminolysis, alcoholy The enzymes of such reactions are widely used in the fields of food, washing, feed, organic synthesis and bioenergy (Curr.Opin.Biotech.2002, 13:390-397). The reaction catalyzed by lipase not only has the characteristics of high selectivity, less side reactions, and high optical purity of the product, but also has the advantages of mild catalytic reaction conditions and en...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12P13/00C12R1/19
CPCC12N9/20C12P13/002C12Y301/01003
Inventor 郑裕国郑仁朝黎小军吴欣玮沈寅初
Owner ZHEJIANG UNIV OF TECH
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