Search

CN-122012446-A - Levan sucrase, mutant, recombinant expression vector and application thereof

CN122012446ACN 122012446 ACN122012446 ACN 122012446ACN-122012446-A

Abstract

The invention discloses levan sucrase, a mutant thereof, a recombinant expression vector and application thereof, and belongs to the technical field of bioengineering. The amino acid sequence of the levansucrase is shown as SEQ ID NO.2 or SEQ ID NO.4, and the amino acid sequence of the levansucrase mutant is shown as SEQ ID NO. 8. A recombinant expression vector comprising a gene encoding levansucrase or a mutant thereof. The levan sucrase and the application of the mutant thereof in the hydrolysis of sucrose or the synthesis of kestose. The levan sucrase has the specific activity of 66.17U/mg, and the levan sucrase mutant with stronger enzyme activity and higher thermal stability is obtained after mutation transformation, and the specific activity of the levan sucrase mutant is 169.37U/mg. The levan sucrase and the mutant thereof can be used for hydrolysis of sucrose and synthesis of kestose, and have good application prospects.

Inventors

  • WANG FEI
  • PENG PEIPEI
  • NI GUORONG
  • YIN XIN
  • ZHOU CHUNHUO
  • HUANG ZHEN
  • LV XIAOJIANG

Assignees

  • 江西农业大学

Dates

Publication Date
20260512
Application Date
20260414

Claims (10)

  1. 1. The levansucrase is characterized in that the amino acid sequence is shown as SEQ ID NO.2 or SEQ ID NO. 4.
  2. 2. The levansucrase-encoding gene of claim 1, wherein the nucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO. 3.
  3. 3. Use of a levansucrase according to claim 1 for the hydrolysis of sucrose or for the synthesis of kestose.
  4. 4. A levan sucrase mutant is characterized in that the amino acid sequence is shown as SEQ ID NO. 8.
  5. 5. The levansucrase mutant encoding gene of claim 4, wherein the nucleotide sequence is shown in SEQ ID NO. 7.
  6. 6. The use of a levansucrase mutant according to claim 4 for the hydrolysis of sucrose or for the synthesis of kestose.
  7. 7. A recombinant expression vector is characterized by comprising a levan sucrase gene shown in SEQ ID NO.3 or a levan sucrase mutant gene shown in SEQ ID NO. 7.
  8. 8. A recombinant engineering bacterium is characterized in that the genome of the recombinant engineering bacterium contains a levan sucrase gene shown as SEQ ID NO.3 or a levan sucrase mutant gene shown as SEQ ID NO.7, and can express levan sucrase shown as SEQ ID NO.4 or a levan sucrase mutant shown as SEQ ID NO. 8.
  9. 9. The recombinant engineering bacterium according to claim 8, wherein the host of the recombinant engineering bacterium is Escherichia coli.
  10. 10. Use of a recombinant engineering bacterium according to claim 8 or 9 for the preparation of a levansucrase according to claim 1 or a levansucrase mutant according to claim 4.

Description

Levan sucrase, mutant, recombinant expression vector and application thereof Technical Field The invention relates to levan sucrase, a mutant thereof, a recombinant expression vector and application thereof, and belongs to the technical field of bioengineering. Background Kestose (CAS number: 470-69-9, molecular formula: C 18H32O16, molecular weight: 504.44) is a non-reducing trisaccharide formed by connecting sucrose with fructose group through beta-1, 2 glycosidic bond, and three isomers of 1-kestose exist, which is the component with the smallest molecular weight and active core in fructo-oligosaccharide (FOS). The kestose has excellent physiological functions, can specifically proliferate probiotics such as bifidobacteria and clostridium prasugrel, maintain intestinal microecological balance, promote T cell differentiation and interferon-gamma secretion, enhance the immunity regulating capability of the organism, and simultaneously have the characteristics of promoting mineral absorption, improving sugar metabolism and the like. As a functional component integrating nutrition and health care functions, the compound feed has wide application prospect in the fields of foods, health care products and medicines, and becomes a hot spot for current prebiotic research. Kestose naturally exists in animals and plants such as onion, jerusalem artichoke, honey and the like, but the content is low and little, and the extraction difficulty is high. Industrially, sucrose is converted by fructosyltransferase. Currently, the biosynthesis of kestose is mainly dependent on sucrose, sucrose 1-fructosyltransferase (1-SST), fructosyltransferase (FTase) and beta-fructofuranosidase (FFase). Among them, 1-SST has higher substrate specificity, is an ideal tool for synthesizing high-purity kestose, but the enzyme extraction difficulty of natural plant sources is high, the cost is high, and the heterogeneous expression system (such as escherichia coli and pichia pastoris) is established, but still faces the bottlenecks of low expression quantity, poor solubility, expensive inducer and the like. Although the cost of the fungus-derived FTase widely used in industry is lower, the problem of insufficient catalytic specificity exists, and byproducts such as kestose, pentasaccharide and the like are easily generated by continuous transglycosylation reaction, so that the separation and purification of target products are difficult. In addition, the existing enzyme preparation has the defects of poor heat stability and acid-base stability, obvious substrate and product inhibition effect and the like, and limits the conversion efficiency under high-concentration substrates, while the immobilization technology can improve reusability, but often has the problems of large mass transfer resistance, low enzyme activity recovery rate and the like, and is difficult to realize industrialized large-scale application. Therefore, development of a kestose synthase with high specificity, high stability and low cost has become a key technical problem to be solved urgently in the current functional oligosaccharide field. Disclosure of Invention Aiming at the prior art, the invention provides levan sucrase, a mutant, a recombinant expression vector and application thereof, and belongs to the technical field of bioengineering. The invention is realized by the following technical scheme: An levansucrase has an amino acid sequence shown as SEQ ID NO.2 or SEQ ID NO. 4. The nucleotide sequence of the coding gene of levansucrase is shown as SEQ ID NO.1 or SEQ ID NO. 3. The levansucrase is applied to the hydrolysis of sucrose and the synthesis of kestose. A levan sucrase mutant has an amino acid sequence shown in SEQ ID NO.8, which is different from levan sucrase shown in SEQ ID NO.4 in that amino acid at position 99 is mutated from valine to isoleucine. The coding gene of the levansucrase mutant has a nucleotide sequence shown in SEQ ID NO.7, and is different from the levansucrase gene shown in SEQ ID NO.3 in that codons at 295-297 locus are mutated from valine codon gtg to isoleucine codon att. The levansucrase mutant is applied to the hydrolysis of sucrose and the synthesis of kestose. A recombinant expression vector contains a levan sucrase gene shown in SEQ ID NO.3 or a levan sucrase mutant gene shown in SEQ ID NO. 7. A recombinant engineering bacterium contains levan sucrase gene shown as SEQ ID NO.3 or levan sucrase mutant gene shown as SEQ ID NO.7 in genome, and can express levan sucrase shown as SEQ ID NO.4 or levan sucrase mutant shown as SEQ ID NO. 8. Further, the host of the recombinant engineering bacteria is escherichia coli (ESCHERICHIA COLI). The recombinant engineering bacteria are applied to the preparation of levan sucrase or levan sucrase mutant. The invention digs a new levan sucrase-MLLase from specific strain, the specific activity of which is 66.17U/mg, has higher enzyme activity, but the thermal stability is not ideal enough. The levan