High-enantioselectivity p-nitrophenyl ethyl esterase mutant as well as construction method and application thereof

Abstract

The invention discloses a p-nitrophenyl ethyl esterase mutant with high enantioselectivity as well as a construction method and application of the p-nitrophenyl ethyl esterase mutant. The p-nitrophenyl ethyl esterase mutant is obtained by mutating an amino acid sequence of wild p-nitrophenyl ethyl esterase pnbA as shown in SEQ ID NO.1 according to one or a combination of more of the following modes: leucine at the 273rd site is mutated into aspartic acid, phenylalanine at the 314th site is mutated into histidine, and leucine at the 362nd site is mutated into arginine. The mutant pnbA-L273D / F314H / L362R provided by the invention shows optimal catalytic performance (E = 47.16) for racemization-acetic acid pinyl hydrate, the enantiomeric excess value of the mutant is 95.13%, the conversion rate of 1S, 5R-acetic acid pinyl hydrate is 90.42%, the enantioselectivity of the mutant is obviously higher than that of a wild type, and the mutant has great application potential in biological catalytic synthesis of monocyclic monoterpenoid chiral alcohols.

Claims

1. A highly enantioselective p-nitrophenylethylesterase mutant, characterized in that: The p-nitrophenylethylesterase mutant is obtained by mutating the amino acid sequence of the wild-type p-nitrophenylethylesterase pnbA as shown in SEQ ID NO.1 in one or more combinations of the following: mutating the leucine at position 273 to aspartic acid, mutating the phenylalanine at position 314 to histidine, and mutating the leucine at position 362 to arginine.

2. The highly enantioselective p-nitrophenylethylesterase mutant according to claim 1, characterized in that The p-nitrophenylethylesterase mutant is obtained by performing combined mutations on the amino acid sequence of the wild-type p-nitrophenylethylesterase pnbA as shown in SEQ ID NO.1 in the following manner: Leucine (L) at position 273 was mutated to aspartic acid (D), phenylalanine (F) at position 314 was mutated to histidine (H), and leucine (L) at position 362 was mutated to arginine (R).

3. The highly enantioselective p-nitrophenylethylesterase mutant according to claim 2, characterized in that The amino acid sequence of the single-site mutant obtained by mutating phenylalanine (F) at position 314 of the amino acid sequence of wild-type p-nitrophenylethylesterase pnbA to histidine (H) is SEQ ID NO. 2; The amino acid sequence of the wild-type p-nitrophenylethylesterase pnbA is mutated from phenylalanine (F) at position 314 to histidine (H) and from leucine (L) at position 362 to arginine (R). The resulting combined mutant has an amino acid sequence of SEQ ID NO.

3. The amino acid sequence of the wild-type p-nitrophenylethylesterase pnbA, in which the leucine (L) at position 273 is mutated to aspartic acid (D), the phenylalanine (F) at position 314 is mutated to histidine (H), and the leucine (L) at position 362 is mutated to arginine (R), yields a combined mutant with the amino acid sequence shown in SEQ ID NO.

4.

4. A gene encoding the p-nitrophenylethylesterase mutant according to any one of claims 1 to 3.

5. The gene according to claim 4, characterized in that The nucleotide sequence encoding the wild-type p-nitrophenylethylesterase pnbA shown in SEQ ID NO.1 is SEQ ID NO.5, and the genes encoding the p-nitrophenylethylesterase mutants are all obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO.

5.

6. A recombinant plasmid comprising the gene according to claim 4 or 5.

7. The recombinant plasmid according to claim 6, characterized in that The plasmid vector of the recombinant plasmid is pET-28a(+).

8. A host cell comprising the gene according to claim 4 or 5 or the recombinant plasmid according to claim 6 or 7.

9. The host cell according to claim 8, characterized in that The host cell is Escherichia coli containing the recombinant plasmid according to claim 6 or 7 E. coli. BL21.

10. A method for constructing a highly enantioselective p-nitrophenylethylesterase mutant according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: culturing the host cell according to claim 8 or 9 to express the p-nitrophenylethyl esterase mutant by induction; collecting the host cells, crushing and centrifuging them, and separating and purifying the p-nitrophenylethyl esterase mutant from the supernatant.

11. A p-nitrophenylethyl esterase mutant according to any one of claims 1 to 3, a gene according to claim 4 or 5, a recombinant plasmid according to claim 6 or 7, or a host cell according to claim 8 or 9 that catalyzes the production of ester compounds before catalysis 1S , 5R -Application of hydrated pinanol.

12. The use according to claim 11, characterized in that The proester compound is racemic-pinene formate hydrate or racemic-pinene acetate hydrate.

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