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CN-122012479-A - Lysine decarboxylase mutant and application thereof in synthesis of 1, 5-pentanediamine

CN122012479ACN 122012479 ACN122012479 ACN 122012479ACN-122012479-A

Abstract

The invention discloses a lysine decarboxylase mutant, a coding gene, a recombinant vector constructed by the coding gene, recombinant genetic engineering bacteria obtained by converting the recombinant vector, and immobilized enzyme prepared by the lysine decarboxylase mutant. The invention also discloses a lysine decarboxylase mutant and application of the immobilized enzyme prepared by the lysine decarboxylase mutant in synthesis of 1, 5-pentanediamine. According to the invention, through site-directed mutagenesis of lysine decarboxylase from different sources, the lysine decarboxylase mutant AfLDC _T88S with high catalytic activity, wider pH tolerance range and good thermal stability is screened out, and is successfully used for catalyzing L-lysine to synthesize 1, 5-pentanediamine, so that the method has a good industrial application prospect.

Inventors

  • WANG ZHEMING
  • TAN HAO

Assignees

  • 杭州因达罗新材料科技有限公司

Dates

Publication Date
20260512
Application Date
20260202

Claims (10)

  1. 1. A lysine decarboxylase mutant is characterized in that the lysine decarboxylase mutant is a mutant of lysine decarboxylase AfLDC, the amino acid sequence of lysine decarboxylase AfLDC is shown as SEQ ID NO. 5, and the lysine decarboxylase mutant is obtained by mutating threonine at position 88 of lysine decarboxylase AfLDC into serine, namely T88S.
  2. 2. A gene encoding the lysine decarboxylase mutant of claim 1.
  3. 3. A recombinant vector constructed from a gene encoding the lysine decarboxylase mutant of claim 2.
  4. 4. A recombinant genetically engineered bacterium obtained by transformation of the recombinant vector of claim 3.
  5. 5. An immobilized enzyme produced by the lysine decarboxylase mutant of claim 1.
  6. 6. The immobilized enzyme according to claim 5, which is prepared by adding a lysine decarboxylase mutant and an amino resin into a phosphate buffer of 50-200mM and pH6.0-8.0, wherein the mass ratio of the lysine decarboxylase mutant to the amino resin is 20-200 mg/1 g, the amino resin comprises LX-1000NH amino resin or ESR-1 amino resin, and 1v/v% -5v/v% glutaraldehyde and 1v/v% -5v/v% polyethylenimine, the immobilization temperature is 4-10 ℃, the stirring speed is 20-100rpm, the immobilization time is 2-5h, and filtering and washing.
  7. 7. The use of the lysine decarboxylase mutant as claimed in claim 1 for synthesizing 1, 5-pentanediamine, wherein the use of the lysine decarboxylase mutant as a catalyst, L-lysine as a substrate, pyridoxal 5-phosphate as a coenzyme, and the enzymatic catalysis of 1, 5-pentanediamine in a reaction medium is carried out under the conditions of temperature and stirring speed control.
  8. 8. The use according to claim 7, characterized in that the reaction medium is an aqueous solution having a pH of 5.0-9.0, the amount of lysine decarboxylase mutants is 20-200mg/L, the concentration of L-lysine is 0.5-2.0M, the concentration of pyridoxal 5-phosphate is 0.05-0.5mM, the temperature is controlled at 40-60 ℃, the stirring speed is 70-300 rpm, and the reaction time is 1-4h.
  9. 9. The use of the immobilized enzyme according to claim 5 or 6 in the synthesis of 1, 5-pentanediamine, characterized in that in the use, the immobilized enzyme is used as a catalyst, L-lysine is used as a substrate, pyridoxal 5-phosphate is used as a coenzyme, and the enzyme catalysis is performed in a reaction medium under the conditions of temperature control and stirring speed to produce 1, 5-pentanediamine.
  10. 10. The use according to claim 9, characterized in that the reaction medium is an aqueous solution having a ph of 5.0-9.0, the amount of immobilized enzyme is 5-20g/L, the L-lysine concentration is 0.5-2.0m, the pyridoxal 5-phosphate concentration is 0.05-0.5mM, the temperature is controlled at 40-60 ℃, the stirring speed is 70-300rpm, and the reaction time is 1-4h.

Description

Lysine decarboxylase mutant and application thereof in synthesis of 1, 5-pentanediamine Technical Field The invention relates to the technical field of enzymatic catalytic synthesis, in particular to a lysine decarboxylase mutant and application thereof in synthesis of 1, 5-pentanediamine. Background 1, 5-Pentanediamine, also known as cadaverine, can be used as a polymer synthesis precursor substance to replace the traditional petroleum-based hexanediamine, and is polycondensed with various dibasic acids to prepare novel bio-based polyamide, and the novel bio-based polyamide material has excellent chemical corrosion resistance, heat resistance and mechanical strength and has great application potential in the industrial fields of packaging, medical equipment, household appliances and the like. At present, the biosynthesis path of 1, 5-pentanediamine is mainly divided into two types, namely a microbial fermentation method and an enzyme catalysis method. Although the microbial fermentation method can realize de novo synthesis, the toxic effect of the product 1, 5-pentanediamine on host cells exists. The enzyme catalysis method is used for efficiently converting the substrate L-lysine into the 1, 5-pentanediamine under specific catalysis conditions by constructing an external reaction system, so that the reaction process is simple, the conditions are easy to control, and the problem of toxicity of the product to cells is avoided. Lysine decarboxylase (Lysine decarboxylase, LDC) is a specific enzyme that catalyzes the decarboxylation of L-lysine to 1, 5-pentanediamine. At present, the research of the enzyme is mostly focused on sources such as escherichia coli, klebsiella, hafnia alvei and the like, but the lysine decarboxylase has the problems of low catalytic activity, poor structural stability and the like in practical application. Therefore, new lysine decarboxylase resources from different strains need to be further excavated, and the catalytic activity and the structural stability of the new lysine decarboxylase are improved by combining enzyme engineering technology so as to promote the industrial application of the new lysine decarboxylase. Disclosure of Invention The invention aims to provide a lysine decarboxylase mutant and application thereof in synthesis of 1, 5-pentanediamine, so as to solve the defects in the prior art. The invention adopts the following technical scheme: The first aspect of the invention provides a lysine decarboxylase mutant, wherein the lysine decarboxylase mutant is a mutant of lysine decarboxylase AfLDC, the amino acid sequence of lysine decarboxylase AfLDC is shown as SEQ ID NO. 5, and the lysine decarboxylase mutant is obtained by mutating threonine at position 88 of lysine decarboxylase AfLDC into serine, namely T88S. In a second aspect, the present invention provides a gene encoding the above lysine decarboxylase mutant. The third aspect of the invention provides a recombinant vector constructed by the coding gene of the lysine decarboxylase mutant. The fourth aspect of the invention provides recombinant genetically engineered bacteria obtained by transformation of the recombinant vector. In a fifth aspect, the present invention provides an immobilized enzyme prepared from the above lysine decarboxylase mutant. Further, the immobilized enzyme is prepared by taking 50-200mM phosphate buffer with pH of 6.0-8.0 as a solvent, adding the lysine decarboxylase mutant and amino resin, wherein the mass ratio of the lysine decarboxylase mutant to the amino resin is 20-200 mg/1 g, the amino resin comprises LX-1000NH amino resin or ESR-1 amino resin, 1v/v% -5v/v% glutaraldehyde and 1v/v% -5v/v% polyethylenimine, the immobilization temperature is 4-10 ℃, the stirring rotation speed is 20-100rpm, the immobilization time is 2-5h, and filtering and washing are carried out to obtain the immobilized enzyme. The sixth aspect of the present invention provides an application of the above lysine decarboxylase mutant in synthesis of 1, 5-pentanediamine, wherein when the lysine decarboxylase mutant is used, the lysine decarboxylase mutant is used as a catalyst, L-lysine is used as a substrate, pyridoxal 5-phosphate is used as a coenzyme, and under the conditions of temperature control and stirring speed, enzyme catalysis is performed in a reaction medium to generate 1, 5-pentanediamine. Further, the reaction medium is aqueous solution with pH of 5.0-9.0, the dosage of lysine decarboxylase mutant is 20-200mg/L, the concentration of L-lysine is 0.5-2.0M, the concentration of pyridoxal 5-phosphate is 0.05-0.5mM, the temperature is controlled to be 40-60 ℃, the stirring speed is 70-300 rpm, and the reaction time is 1-4h. The seventh aspect of the present invention provides an application of the immobilized enzyme in synthesis of 1, 5-pentanediamine, wherein the immobilized enzyme is used as a catalyst, L-lysine is used as a substrate, pyridoxal 5-phosphate is used as a coenzyme, and the immobilized