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CN-122012427-A - KpADH alcohol dehydrogenase mutant and application thereof in syringol synthesis

CN122012427ACN 122012427 ACN122012427 ACN 122012427ACN-122012427-A

Abstract

The invention provides KpADH alcohol dehydrogenase mutant and application thereof in synthesis of eugenol, wherein under the condition that a recombinant expression vector pET28a-KpADH is taken as a template, single-point or double mutation is carried out on 165 th and 231 th positions of KpADH alcohol dehydrogenase with an amino acid sequence shown as SEQ ID NO.1, so as to obtain KpADH alcohol dehydrogenase mutant. The mutant exhibits enhanced catalytic rates. Wherein, the KpADH alcohol dehydrogenase mutant C165F-V231D whole-cell catalyst reduces 20mM syringaldehyde into eugenol within 24 hours, and the yield reaches 92.1 percent.

Inventors

  • HE YUCAI
  • LIN RUN
  • LI XIN
  • Chai Haoyu
  • FAN BO

Assignees

  • 常州大学

Dates

Publication Date
20260512
Application Date
20260211

Claims (10)

  1. 1. An alcohol dehydrogenase mutant, which is characterized in that the mutant is obtained by mutating an amino acid sequence shown as SEQ ID NO.1, wherein the mutation is one or more of the following mutations: (1) Cysteine 165 mutating to phenylalanine; (2) Valine at position 231 was mutated to aspartic acid.
  2. 2. The alcohol dehydrogenase mutant according to claim 1, wherein the amino acid sequence of the alcohol dehydrogenase mutant is shown in SEQ ID NO. 2-4.
  3. 3. A coding gene, characterized in that it codes for the alcohol dehydrogenase mutant according to claim 1 or 2.
  4. 4. The coding gene according to claim 3, wherein the alcohol dehydrogenase mutant according to claim 2 is encoded, and the nucleotide sequence of the alcohol dehydrogenase mutant is shown in SEQ ID NO. 6-8.
  5. 5. A recombinant vector comprising the coding gene according to claim 3 or 4.
  6. 6. An expression strain comprising the coding gene of claim 3 or 4, or the recombinant vector of claim 5.
  7. 7. The application of the alcohol dehydrogenase in synthesizing the eugenol by enzyme catalysis of syringaldehyde is characterized in that the amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID NO. 1-4.
  8. 8. The use according to claim 7, wherein the alcohol dehydrogenase is added to the enzyme catalytic system in the form of whole cells of the expression strain.
  9. 9. The use according to claim 7, wherein the enzyme catalytic system further comprises syringaldehyde, glucose and NADPH, wherein the concentration of syringaldehyde is 20 mM, and the concentration of whole cells of the expression strain is 0.05g/mL.
  10. 10. The use according to claim 9, characterized in that the molar ratio of syringaldehyde, glucose, NADPH is 20:40:0.002.

Description

KpADH alcohol dehydrogenase mutant and application thereof in syringol synthesis Technical Field The invention relates to the technical fields of enzyme catalysis engineering and protein modification engineering, in particular to KpADH alcohol dehydrogenase mutant and application thereof in syringol synthesis. Background Eugenol is an important chemical intermediate and has wide application in perfumery and pharmaceutical production. The eugenol has remarkable inhibiting effect on various bacteria and fungi clinically, can destroy the cell structure of microorganisms or interfere the metabolism of the microorganisms, reduces the breeding of pathogens, and is commonly used for assisting in treating infectious diseases caused by the bacteria or the fungi. It can be obtained by reduction of syringaldehyde, a compound derived from lignin. As the yield of plant extraction is low, panyadee et al offer a promising alternative to the use of microbial bioconversion for the preparation of eugenol. Putra et al utilized the strain DALE-22 to catalyze the efficient conversion of 22.3 mM syringaldehyde to 18.7 mM eugenol, highlighting the advantages of the biosynthetic route in terms of mild reaction conditions and environmental sustainability. However, the cells have a complexity and require a consistent control of fermentation conditions in a short period of time. KpADH is the identification of an NADPH alcohol dehydrogenase from KP, recombinant E.coli KPADH contains Kluyveromyces polysporus reductase (ADH) and Bacillus subtilis Glucose Dehydrogenase (GDH), and in recent years GDH has been able to biologically convert NAD (P) + to NAD (P) H, while co-substrate glucose oxidizes to regenerate coenzyme, ADH coupled with Glucose Dehydrogenase (GDH) for coenzyme regeneration has been effectively used to prepare biobased alcohols. The synthesis of (S) -N-Boc-3-hydroxypiperidine ((S) -NBHP) from the beginning by catalytic reduction with wild-type alcohol dehydrogenase KpADH has low catalytic activity and cannot be adapted to industrial production. Disclosure of Invention In view of the above, the present invention aims to provide KpADH alcohol dehydrogenase mutants and their use in syringol synthesis. The invention firstly provides a wild type alcohol dehydrogenase which is alcohol dehydrogenase KpADH derived from Kluyveromyces (Kluyveromyces polyspor), the amino acid sequence of which is shown as SEQ ID NO.1, and the wild type alcohol dehydrogenase shows excellent catalytic activity when reducing a large-volume ketone substrate. Site-directed mutagenesis is carried out on the amino acid sequence of the wild-type alcohol dehydrogenase to obtain an alcohol dehydrogenase mutant, wherein the mutation comprises one or more of the following mutations: (1) Mutating cysteine (C) at position 165 to phenylalanine (F); (2) Valine (V) at position 231 was mutated to aspartic acid (D). Preferably, the 165 th site of the amino acid sequence of the wild-type alcohol dehydrogenase is mutated into F and the 231 st site is mutated into D to obtain a mutant C165F-V231D, the amino acid sequence of which is shown as SEQ ID NO.2, or the 165 th site of the amino acid sequence of the wild-type alcohol dehydrogenase is mutated into F, the amino acid sequence of which is shown as SEQ ID NO.3, and the 231 st site of the amino acid sequence of the wild-type alcohol dehydrogenase is mutated into D to obtain a mutant V231D, the amino acid sequence of which is shown as SEQ ID NO. 4. The invention further provides genes encoding the above wild-type alcohol dehydrogenase and alcohol dehydrogenase mutants. Preferably, the nucleotide sequence of the wild alcohol dehydrogenase is shown as SEQ ID NO.5, and the nucleotide sequence of the alcohol dehydrogenase mutant is shown as SEQ ID NO. 6-8. The invention further provides a recombinant vector containing the coding gene and an expression strain containing the coding gene or the recombinant vector. The preparation method of the recombinant vector and the expression strain comprises the steps of connecting the nucleotide sequence of the alcohol dehydrogenase KpADH mutant to a pET vector through an enzyme cutting site to obtain the recombinant vector, and converting the recombinant vector to an expression system through a heat shock method to obtain the mutant expression strain. The invention also provides application of the wild alcohol dehydrogenase and the alcohol dehydrogenase mutant in catalyzing substrate syringaldehyde to synthesize eugenol. In particular, the enzyme catalytic system comprises a substrate syringaldehyde, a co-substrate glucose, NADPH and an alcohol dehydrogenase. Further, the alcohol dehydrogenase is added in the form of wet bacteria prepared by high-density fermentation of the expression strain. Further, in the enzyme catalysis system, the concentration of syringaldehyde is 20mM, and the concentration of wet thalli is 0.05 g/mL. In the enzyme catalysis system, the molar ratio of syringaldeh