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Thermostability modification of a maleate cis-trans isomerase and its application

A technology of cis-trans isomerization and maleic acid, applied in the field of enzyme engineering, can solve the problems of loss and decrease of enzyme activity

Active Publication Date: 2019-06-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The modification of the enzyme active center often results in a decrease or loss of enzyme activity

Method used

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  • Thermostability modification of a maleate cis-trans isomerase and its application
  • Thermostability modification of a maleate cis-trans isomerase and its application
  • Thermostability modification of a maleate cis-trans isomerase and its application

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Embodiment 1

[0021] According to the amino acid sequence of the wild-type maleate cis-trans isomerase shown in SEQ ID NO.1, the maleate cis-trans isomerase mutant primers are designed as shown in Table 1, with pET-24a (+)- maiA (construction of the plasmid, see literature [Wang Ya, Cui Wenjing, Zhou Li, Liu Zhongmei, Zhou Zhemin. Expression, purification and enzymatic properties of Serratia marcescens maleate cis-trans isomerase [J]. Food and Biology Technical Journal 2014, Volume 33, No. 11, 1204-1209]) was used as a template for full-plasmid PCR to achieve site-directed mutation. Proceed as follows:

[0022] (1) Semi-rational design to select amino acids to be mutated and design mutation primers;

[0023] (2) Carry out whole plasmid PCR with pET-24a(+)-maiA as template to realize site-directed mutation (such as figure 1 );

[0024] (3) PCR product was digested overnight with DpnI enzyme;

[0025] (4) The digested product is transformed into the competent cells of JM109, and then appl...

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Abstract

The invention discloses thermostability transformation of maleic acid cis-trans isomerase and application thereof, and belongs to the field of enzyme engineering. The maleic acid cis-trans isomerase gene maiA from serratia marcescens is subjected to thermostability transformation by a semi-rational design method. Compared with the wild maleic acid cis-trans isomerase, the obtained mutant has the advantages that the half-life period of the mutant G27A-G171A at 55 DEG C is 2.9 times of that of the wild type, and the enzyme activity is 1.96 times of that of the wild type; the half-life period of the mutant G27A-K104R is 4.18 times of the wild type at 55 DEG C, and the enzyme activity is 1.59 times of that of the wild type.

Description

technical field [0001] The invention relates to thermal stability modification and application of maleate cis-trans isomerase, which belongs to the field of enzyme engineering. Background technique [0002] Maleate cis-trans isomerase catalyzes the conversion of maleic acid into fumaric acid, and fumaric acid can be converted into aspartic acid with higher added value through aspartase or into apple through fumarase acid, in order to realize the direct conversion from maleic acid to aspartic acid or malic acid without the separation and purification of the intermediate product fumaric acid, it is necessary to combine maleic acid cis-trans isomerase with aspartase or Fumarase is used in combination. Regardless of whether it is aspartase or fumarase, its thermal stability is higher than that of maleate cis-trans isomerase. In order to realize the continuity and reusability of this coupled catalytic system, horses are required. Toic acid cis-trans isomerase has good activity ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12P7/46C12P13/20C12R1/19
CPCC12N9/90C12P7/46C12P13/20C12Y502/01001
Inventor 周哲敏余龙周丽崔文璟刘中美郭军玲张斌
Owner JIANGNAN UNIV
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