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CN-120966800-B - Aminopeptidase mutant and encoding gene, recombinant vector, recombinant strain and application thereof

CN120966800BCN 120966800 BCN120966800 BCN 120966800BCN-120966800-B

Abstract

The invention relates to the field of enzyme engineering, and discloses an aminopeptidase mutant, a coding gene, a recombinant vector, a recombinant strain and application thereof, wherein the aminopeptidase mutant is obtained by mutating at least one of S88L, Y C and Q268F of an enzyme with an amino acid sequence shown as SEQ ID NO. 1. The aminopeptidase mutant disclosed by the invention can catalyze L-histidine to synthesize L-carnosine with high selectivity in the presence of beta-alanine methyl ester, has higher L-carnosine yield and lower impurity content, and has industrial application value.

Inventors

  • LI JIACHENG
  • YANG JIANHUA
  • BIAN YUHUA
  • YU HONGZHONG

Assignees

  • 上海昱菘生物科技有限公司

Dates

Publication Date
20260508
Application Date
20251020

Claims (10)

  1. 1. An aminopeptidase mutant is characterized in that the aminopeptidase mutant is obtained by mutating an enzyme with an amino acid sequence shown as SEQ ID NO. 1 with any one of S88L, Y C and Q268F.
  2. 2. The aminopeptidase mutant according to claim 1, characterized in that the aminopeptidase mutant is an amino acid sequence shown in SEQ ID No. 2, an amino acid sequence shown in SEQ ID No. 3 or an amino acid sequence shown in SEQ ID No. 4.
  3. 3. A gene encoding an aminopeptidase mutant, wherein the gene encoding an aminopeptidase mutant comprises a nucleotide sequence encoding the aminopeptidase mutant of claim 1 or 2.
  4. 4. The gene according to claim 3, wherein the gene encoding the aminopeptidase mutant is a nucleotide sequence shown as SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8.
  5. 5. A recombinant vector comprising the gene of claim 3 or 4.
  6. 6. A recombinant strain comprising the gene of claim 3 or 4 or the recombinant vector of claim 5.
  7. 7. Use of at least one of the aminopeptidase mutant of claim 1 or 2, the gene of claim 3 or 4, the recombinant vector of claim 5, the recombinant strain of claim 6 for the synthesis of L-carnosine.
  8. 8. A method for synthesizing L-carnosine, comprising catalyzing the synthesis of L-carnosine by L-histidine in the presence of beta-alanine methyl ester or a salt thereof and the aminopeptidase mutant of claim 1 or 2.
  9. 9. The method according to claim 8, wherein the content of beta-alanine methyl ester or salt thereof in the catalytic system is 50-200g/L, and the content of L-histidine is 40-80 mmol/L; the catalysis conditions comprise pH of 7-10 and temperature of 20-50 ℃.
  10. 10. The method according to claim 9, characterized in that beta-alanine methyl ester or a salt thereof and/or L-histidine are added to the catalytic system in a batch-wise manner.

Description

Aminopeptidase mutant and encoding gene, recombinant vector, recombinant strain and application thereof Technical Field The invention relates to the field of enzyme engineering, in particular to an aminopeptidase mutant, a gene for encoding the aminopeptidase mutant, a recombinant vector, a recombinant strain, application of at least one of the aminopeptidase mutant, the gene, the recombinant vector and the recombinant strain in synthesis of L-carnosine, and a method for synthesizing L-carnosine. Background L-carnosine is also called beta-alanyl-L-histidine, is a dipeptide obtained by condensing two amino acids of beta-alanine and L-histidine, and is a natural antioxidant existing in organisms. Carnosine is a natural dipeptide that is found in large quantities in mammalian muscles and brain. In vivo, carnosine has pH buffering, antioxidant, free radical resisting, and antiaging effects. Clinically, the zinc-containing carnosine can be used for relieving visual fatigue and treating cataract, and can be used for treating gastric ulcer. Compared with other antioxidants, the L-carnosine has the advantages of strong antioxidant capacity, no toxic or side effect, various physiological activities and wide application prospect in the fields of medicine, health care, sanitation, beauty and the like. There are various methods for producing L-carnosine. Because of the high content of carnosine in animal muscle, the early extraction of carnosine from animal tissue is carried out directly, and the method has low yield and poor purity and has been eliminated. In the traditional chemical synthesis method, active groups of L-histidine and beta-alanine are respectively activated and protected, and protective groups are eliminated after subsequent condensation reaction, so that the method has high yield, but the whole synthesis process has a plurality of reaction steps, and highly toxic preparations such as hydrazine hydrate and the like are needed, and the residual hydrazine is not allowed in the L-carnosine product, so that the high requirements on the extraction and refining of the product are met. Various enzymatic synthesis methods have been reported, including reverse hydrolysis synthesis of beta-alanine and L-histidine catalyzed by dipeptidyl hydrolase, synthesis of beta-alaninamide/alanine ester and L-histidine catalyzed by aminopeptidase, and condensation reaction synthesis of ATP-powered beta-alanine and L-histidine catalyzed by carnosine synthetase. Wherein the reaction catalyzed by the dipeptidyl hydrolase is limited by the reaction balance, high-concentration beta-alanine is required to be added for reaction, and the conversion rate of L-histidine is only about 30 percent at maximum. Whereas the carnosine synthase-catalyzed reaction relies on ATP, there is a cost problem. Compared with the two types of enzymatic reactions, the aminopeptidase catalytic reaction does not need to add high-concentration histidine, does not depend on ATP, has fewer reaction steps, and is more suitable for industrial application. However, the aminopeptidase reported at present has low activity, a plurality of byproducts and complex product separation and purification, which severely limits the practical application of the method, so that the aminopeptidase with high activity and high selectivity is necessary to be developed. Disclosure of Invention The invention aims to provide aminopeptidase with high activity and high selectivity, in particular to aminopeptidase and encoding genes, recombinant vectors, recombinant strains and application thereof, the aminopeptidase can catalyze L-histidine to synthesize L-carnosine with high selectivity in the presence of beta-alanine methyl ester, has high yield and has industrial application value. In order to achieve the above object, the first aspect of the present invention provides an aminopeptidase mutant obtained by mutating an enzyme having an amino acid sequence shown in SEQ ID NO. 1 with at least one of S88L, Y C and Q268F. Preferably, the aminopeptidase mutant is an amino acid sequence shown in SEQ ID NO. 2, an amino acid sequence shown in SEQ ID NO. 3 or an amino acid sequence shown in SEQ ID NO. 4. In a second aspect the invention provides a gene encoding an aminopeptidase mutant comprising a nucleotide sequence encoding an aminopeptidase mutant as described above. Preferably, the gene encoding the aminopeptidase mutant is a gene having a nucleotide sequence shown in SEQ ID NO. 6, SEQ ID NO. 7 or SEQ ID NO. 8. In a third aspect, the present invention provides a recombinant vector comprising a gene as described above. In a fourth aspect the present invention provides a recombinant strain comprising a gene as described above or a recombinant vector as described above. In a fifth aspect, the invention provides the use of at least one of an aminopeptidase mutant as described above, a gene as described above, a recombinant vector as described above, and a recombinant strain as described abo