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CN-121975781-A - Mannose isomerase mutant with improved enzyme activity and application thereof

CN121975781ACN 121975781 ACN121975781 ACN 121975781ACN-121975781-A

Abstract

The invention relates to a mannose isomerase mutant with improved enzyme activity and application thereof, wherein the amino acid sequence of the mannose isomerase mutant is shown as SEQ ID NO.4, the invention carries out molecular modification on the mannose isomerase from Stenotrophomonas maltophilia, the proline at 10 th position is subjected to site-directed mutagenesis to form alanine, the methionine at 139 th position is subjected to site-directed mutagenesis to form isoleucine to obtain mutant P10A+M139I, and pure enzyme is obtained after purification by a nickel affinity chromatographic column, the optimal pH value is 8.5, the optimal temperature is 60 ℃, the relative enzyme activity is improved by 28.2% compared with that of wild mannose, an excellent catalyst is provided for mannose production in industry, the production cost of mannose is reduced, and a foundation is laid for the research of mannose isomerase and mannose.

Inventors

  • Lv Yizheng
  • CHEN ZIYI
  • NI DAWEI
  • ZHANG WENLI
  • MU WANMENG

Assignees

  • 江南大学

Dates

Publication Date
20260505
Application Date
20260224

Claims (10)

  1. 1. The mannose isomerase mutant with improved enzyme activity is characterized in that the amino acid sequence of the mannose isomerase mutant is shown as SEQ ID NO. 4.
  2. 2. The mannose isomerase mutant with improved enzyme activity according to claim 1, wherein the mannose isomerase has an amino acid sequence shown in SEQ ID No.2, in which proline at position 10 is site-directed mutated to alanine and methionine at position 139 is site-directed mutated to isoleucine.
  3. 3. The mannose isomerase mutant with improved enzyme activity according to claim 2, wherein the mannose isomerase with the amino acid sequence shown in SEQ ID No.2 is derived from Stenotrophomonas maltophilia.
  4. 4. A gene encoding the mannose isomerase mutant of claim 1.
  5. 5. A recombinant plasmid carrying the gene of claim 4.
  6. 6. The recombinant plasmid according to claim 5, wherein the expression vector of the recombinant plasmid is PET-28a (+).
  7. 7. A recombinant cell expressing the mannose isomerase mutant of claim 1 or the gene of claim 2, wherein the recombinant plasmid of claim 5 is obtained by transforming a host cell comprising a prokaryotic cell or a eukaryotic cell.
  8. 8. A method for producing the mannose isomerase mutant according to claim 1, wherein the recombinant cell according to claim 7 is subjected to induction culture to produce the mannose isomerase mutant.
  9. 9. Use of a mannose isomerase mutant as claimed in claim 1 for the preparation of mannose.
  10. 10. The use according to claim 9, wherein the substrate D-fructose is enzymatically reacted to mannose under the action of a mannose isomerase mutant, wherein the pH of the enzymatic reaction system is 8.5, the temperature of the enzymatic reaction is 60 ℃, and the reaction time of the enzymatic reaction is 5min.

Description

Mannose isomerase mutant with improved enzyme activity and application thereof Technical Field The invention belongs to the technical field of genetic engineering and fermentation of enzymes, and particularly relates to a mannose isomerase mutant with improved enzyme activity and application thereof. Background D-mannose (D-Mannose) is a naturally occurring rare aldohexose with a sweetness of about 60% of sucrose and 86% of glucose, and has a caloric value of only 3.75 kcal/g and exhibits remarkable physiological functions including blood sugar lowering, urinary tract infection prevention, antitumor, and cancer treatment. D-mannose has a wide range of uses in the food industry, in the nutritional and pharmaceutical industries, and has also been proposed as a carbohydrate nutrient supplement for infants and children, with great potential for use. However, D-mannose is very low in natural abundance (only in part of fruits, microorganisms, animal body fluids and tissues), and large-scale production is difficult to achieve by conventional extraction methods. At present, the industrialized production of D-mannose mainly depends on a plant extraction method, a chemical method and a biological method. Plant extraction methods include acid hydrolysis (sulfuric acid treatment), enzymatic hydrolysis and microbial fermentation. The raw materials are widely available, such as litchi rind, apple pulp and the like, and D-mannose can be obtained by adding ethanol for precipitation separation after sulfuric acid hydrolysis. However, the method uses a large amount of sulfuric acid and ethanol, has the problems of environmental pollution risk, health threat of operators, high cost caused by raw material dependence and the like, and severely restricts the large-scale application of the method. The chemical method takes D-glucose as a raw material and takes molybdate as a catalyst for catalysis at the temperature of 150 ℃ and the pH value of 3.0, but the method has the problems of high production cost, high energy consumption, harsh reaction conditions and the like, and the biological method realizes high-efficiency conversion by enzyme catalysis, and key enzymes comprise D-mannose isomerase (D-MIase), D-mannose epimerase (D-MEase) and the like. D-MIase catalyzes the isomerization of D-fructose to D-mannose, and the conversion rate is stabilized at about 25%. Wherein, D-MIase becomes an ideal choice for large-scale production of D-mannose by virtue of high conversion rate and industrial applicability. However, the currently known natural D-MIase has the defects of low enzyme activity, poor heat stability and the like, so that the industrial application is hindered. Although researchers try to reform D-MIase through directed evolution and rational design, the technical bottlenecks of low substrate conversion rate, insufficient industrial-grade enzyme activity, poor long-term heat stability and the like are not broken through. Therefore, mannose Isomerase (MI) is screened and directionally transformed through directed evolution and rational design, so that mannose isomerase mutants with improved enzyme activity and good thermal stability are obtained, and the method has important significance for industrial production of mannose. Disclosure of Invention The invention aims to overcome the defects in the prior art, provides a mannose isomerase mutant with improved enzymatic activity and application thereof, has the optimal temperature of enzymatic reaction of 60 ℃, has good thermal stability, and has improved enzymatic activity compared with wild mannose isomerase to a certain extent, thereby laying a solid foundation for industrialized application of D-mannose and mannose isomerase. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: A mannose isomerase mutant with improved enzyme activity, wherein the amino acid sequence of the mannose isomerase mutant is shown as SEQ ID NO. 4. As a further technical scheme, the mannose isomerase is obtained by site-directed mutagenesis of proline at the 10 th position of mannose isomerase with an amino acid sequence shown as SEQ ID No.2 into alanine and site-directed mutagenesis of methionine at the 139 th position into isoleucine. As a further technical scheme, the mannose isomerase with the amino acid sequence shown in SEQ ID No.2 is derived from Stenotrophomonas maltophilia. A gene encoding the mannose isomerase mutant. A recombinant plasmid carrying the gene. As a further technical scheme, the expression vector of the recombinant plasmid is PET-28a (+). A recombinant cell expressing the mannose isomerase mutant or the gene, and transforming the recombinant plasmid into a host cell, wherein the host cell comprises a prokaryotic cell or a eukaryotic cell. A method for preparing the mannose isomerase mutant, which is to perform induction culture on the recombinant cells to produce the mannose isomerase mutant. The method comprises the steps of ob