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Lipase mutant with improved thermal stability

A technology of thermal stability and lipase, applied in the field of genetic engineering, can solve problems such as poor thermal stability

Active Publication Date: 2018-06-05
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] For the above deficiencies or improvement needs of the prior art, the invention provides a lipase mutant with improved thermostability, which is obtained from the parent Candida rugosa (ATCC NO: 14830) lipase LIP1 (Candida Rugosa Lipase 1, CRL1) Perform site-directed mutagenesis to obtain a lipase mutant with improved thermal stability. The parental CRL1 amino acid sequence is mutated at the amino acid substitution position, and "original amino acid / position / replacement amino acid" is used to represent the mutated amino acid in the lipase mutant. The fat The enzyme mutant is Asp457Phe to solve the technical problem of poor thermal stability of Candida rugosa lipase LIP1 (CRL1) in the prior art

Method used

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  • Lipase mutant with improved thermal stability
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  • Lipase mutant with improved thermal stability

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Embodiment 1

[0053] (1) Using the parental gene as a template, the gene encoding the lipase mutant was cloned by the whole plasmid PCR method.

[0054] The present inventors designed mutation primers using the gene sequence of Candida rugosa lipase LIP1 (CRL1) as a template, and obtained the lipase mutant gene by PCR.

[0055] Design primers:

[0056] Primer 1:

[0057] F chain: (SEQ ID: NO4)

[0058] 5'-CCGCTCGAGCATCATCACCATCACCACGCCCCTACTGCTACTCTT-3'

[0059] R chain: (SEQ ID: NO5)

[0060] 5'-TTGCGGCCGCTTAGACAAAGAAAGAAGGTGGGTTACTAAAAAGAGCG-3'

[0061] Using the above primers, use the parental lipase gene shown in SEQ ID: NO 3 as a template to amplify the CRL1 whole gene, and simultaneously introduce EcoR I and Not I restriction sites at both ends, and the PCR amplification system is as follows:

[0062]

[0063] The PCR reaction program was: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 30 s, annealing at 68°C for 30 s, extension at 72°C for 1 min and 30 s, and 30...

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Abstract

The present invention provides a Candida rugosa lipase mutant, wherein Candida rugosa (ATCC NO:14830) lipase LIP1 (CRL1) is subjected to thermal stability modification by using a site-directed mutation technology so as to optimize the practicality in the high temperature industrial environment. According to the present invention, the CRL1 gene is used as the template, the protein rational design technology is used, and the amino acid Asp at 4578 site of the CRL1 lipase gene sequence is substituted with Phe so as to obtain the lipase mutant with the improved thermal stability, wherein the application range and the catalytic efficiency of the enzyme are improved; and the CRL1 is subjected to site-directed mutation by using the molecular biology method to obtain the lipase mutant, wherein theTm value of the lipase mutant is increased by 9.4 DEG C, the optimum temperature is increased by 10 DEG C, and the T1 / 2 (50 DEG C) is 6.5 times the T1 / 2 (50 DEG C) of the parent lipase.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a lipase mutant with improved thermostability and a lipase gene. Background technique [0002] As a natural biocatalyst, enzyme has the incomparable advantages of mild reaction conditions, high catalytic efficiency, and high specificity of substrate selection, which cannot be compared with traditional catalytic methods. It has great application potential and good development prospects in industrial production. Production often requires a higher reaction temperature. On the one hand, the reaction under high temperature conditions can significantly reduce the probability of the reactant being contaminated by microorganisms, and at the same time increase the solubility of the substrate, making the substrate more accessible to the enzyme; on the other hand On the other hand, in general, every 10°C increase in the reaction temperature will double the reaction rate. ...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N15/11
CPCC12N9/20C12Y301/01003
Inventor 闫云君李冠霖苏枫陈苑方兴容徐莉
Owner HUAZHONG UNIV OF SCI & TECH
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