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Method for improving structural isomerism catalytic activity of CE enzyme and mutant thereof

A mutant and epimerase technology, applied in the field of enzyme engineering, can solve the problem of poor substrate affinity, which restricts the process of industrial application, and the catalytic properties of substrate affinity, lactulose conversion rate and thermal stability have not been greatly improved and other issues to achieve good catalytic performance

Active Publication Date: 2021-10-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the method of using CE enzyme to prepare lactulose still has poor substrate affinity, low structural isomerization activity (no more than 10% of the epimerization activity in most cases), and the generation of by-product ipilactose, etc. Defects restrict its industrial application process
So far, many achievements have been made in the transformation of CsCE, such as improving its structural isomerism activity through random mutation method; improving the thermal stability of CE enzyme through site-directed mutation method, etc., but its substrate affinity, lactulose conversion rate and thermal Catalytic properties such as stability have not been greatly improved

Method used

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  • Method for improving structural isomerism catalytic activity of CE enzyme and mutant thereof
  • Method for improving structural isomerism catalytic activity of CE enzyme and mutant thereof
  • Method for improving structural isomerism catalytic activity of CE enzyme and mutant thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Determining the substrate binding site of CsCE and constructing a mutant plasmid

[0040] (1) Comparative analysis of amino acid sequence and crystal structure to determine the mutation site

[0041] like figure 1 As shown in the crystal structure comparison diagram and amino acid sequence comparison, the two tryptophan (Tryptophan, W) in the active center of CE enzyme can recognize and fix the disaccharide substrate, and the tryptophan W308 near the reducing end of the substrate is in the N - The acetyl-glucosamine superfamily is conserved, while the non-reducing end tryptophan W372 is only conserved in the CE enzyme family. Therefore, it is speculated that the W372 site affects the activity of CE enzymes in catalyzing disaccharides.

[0042] (2) Construction of wild-type recombinant plasmid pET-28b-CsCE

[0043] ① The whole genome of Caldicellulosiruptorsaccharolyticus DSM 8903 was extracted as a template, primers were designed according to the CsCE gene s...

Embodiment 2

[0071] Example 2: Remodeling the substrate binding pocket and constructing mutants based on semi-rational design

[0072] In order to improve the structural isomerization activity of CsCE, a semi-rational design strategy was used to perform saturation mutations on the amino acids around the substrate-binding related sites CsCE-W308 and CsCE-W372. Sequence alignment and crystal structure analysis of CE family enzymes were carried out, and two residues near tryptophan and at the entrance of the substrate were selected for molecular modification ( figure 2 ), respectively CsCE-I306 and CsCE-W307 near the substrate reducing end CsCE-W308 (divided into A region); CsCE-Q371 near the substrate non-reducing end CsCE-W372 (divided into B region); CsCE active center At the entrance of CsCE-W355 (divided into C region), the PCR method was used to perform saturation mutation on these 4 sites.

[0073] The PCR amplification reaction system and reaction amplification conditions were the s...

Embodiment 3

[0077] Example 3: Study on Enzymatic Properties of Cellobiose Epimerase

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Abstract

The invention discloses a method for improving the structural isomerism catalytic activity of a CE enzyme and a mutant thereof, and belongs to the field of enzyme engineering. According to the invention, cellobiose epimerase (CsCE) derived from Caldicellosirutorschcharlyticus is taken as a research object, site-saturation mutagenesis is carried out on a site related to substrate binding based on a semi-rational strategy of sequence alignment and crystal structure analysis, a plurality of mutants with obviously improved structural isomerism catalytic activity are obtained, and the structural isomerism activity of the mutants is improved by about 36-232% than that of a wild type CE enzyme. The invention provides a feasible scheme for CE enzyme improvement, and has important significance for promoting enzymatic industrial synthesis of lactulose.

Description

technical field [0001] The invention relates to a method for improving the catalytic activity of CE enzyme structural isomerism and a mutant thereof, belonging to the field of enzyme engineering. Background technique [0002] Lactulose (Lactulose, β-D-galactosyl-1,4-D-fructose) is a non-digestible disaccharide, because of its ability to promote the growth of bifidobacteria, regulate intestinal pH, and inhibit the growth of pathogenic bacteria It is widely used in the food and pharmaceutical industries due to its prebiotic properties. At present, commercial lactulose on the market is mainly produced by chemical methods, which will produce high levels of by-products, and chemical methods involve complicated separation and purification steps, which will easily cause serious environmental pollution. These defects restrict the production of lactulose. application. Enzymatic synthesis of lactulose has become a research hotspot in the current production of lactulose because of it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/70C12N1/21C12P19/24C12P19/12A23L29/00A61K48/00A61K38/52A61P1/00
CPCC12N9/90C12N15/70C12P19/24C12P19/12C12Y501/03011A23L29/06A23L29/065A61K48/005A61K38/52A61P1/00
Inventor 吕小妹王璐杨瑞金
Owner JIANGNAN UNIV
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