Aspergillus usamii epoxide hydrolase mutants with improved enantioselectivity

A technology of Aspergillus Usami and epoxides, applied in the field of enzyme engineering and biocatalysis, can solve the problems of low enantioselectivity and limited application potential

Active Publication Date: 2016-10-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enantioselectivity of the recombinant enzyme to epoxides is not high, which limits its application potential in the chiral synthesis of high value-added prodrugs

Method used

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  • Aspergillus usamii epoxide hydrolase mutants with improved enantioselectivity
  • Aspergillus usamii epoxide hydrolase mutants with improved enantioselectivity
  • Aspergillus usamii epoxide hydrolase mutants with improved enantioselectivity

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Experimental program
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Effect test

Embodiment 1

[0019] Example 1: Implementation of site-specific saturation mutation

[0020] (1) Using the construction method of recombinant plasmid pET-28a(+)-Aueh2 (see the patent application of Patent Publication No. CN102994470A) as template, A250X-F and pET28-R as primers, using PrimeSTAR DNA polymerase (purchased from TaKaRa) to carry out The first round of PCR amplification (95°C 4min; 98°C 10s, 55°C 5s, 72°C 3.5min, 30 cycles; 72°C 10min) obtained a large primer A250X-1st; using the large primer A250X-1st as a primer, recombinant The plasmid pET-28a(+)-Aueh2 was used as a template for the second round of PCR amplification (95°C for 4min; 98°C for 10s, 55°C for 10s, 72°C for 3.5min, 25 cycles; 72°C for 10min); A250X-2st PCR product Transform E.coli BL21(DE3) competent cells after digesting (37°C, 2h) the template pET-28a(+)-Aueh2 with Dpn I, coat kanamycin-resistant LB plates and culture at 37°C for 12-16h to obtain Recombinant library.

[0021] A250X-F: TTTGGCAGTGGTTAC GTCGAGCA...

Embodiment 2

[0027] Example 2: Determination of Enantioselectivity of Recombinant AuEH2 and AuEH2 Mutant Enzyme

[0028]Add 500 μL of bacterial suspension and 450 μL of sodium phosphate buffer (pH 7.5) to a 1.5 mL EP tube, incubate at 10°C for 5 min, then add 50 μL of rac-SO (final concentration 10 mmol / L) for reaction. 50 μL of samples were regularly extracted to 1 mL of ethyl acetate (containing 1 mmol / L n-hexanol as an internal standard) for extraction, and the samples were analyzed using a gas chromatograph GC-2010 (Shimadzu, Japan), a chiral gas chromatography column, and a hydrogen flame ionization detector. The analysis conditions were: inlet and detector temperature 250°C; initial column temperature 100°C, rising to 195°C at 5°C / min; carrier gas nitrogen, flow rate 3.0mL / min, split ratio 1:50. The retention times of n-hexanol, (R)-styrene oxide and (S)-styrene oxide were 3.477, 5.959 and 6.065 min, respectively. Substrate e.e. s =[(S-R) / (R+S)]×100%; E=ln[(1-c)×(1-e.e. s )] / ln[(1...

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Abstract

The invention discloses aspergillus usamii epoxide hydrolase mutants with improved enantioselectivity, and belongs to the technical field of enzyme engineering and biological catalysis. Aspergillus usamii epoxide hydrolases (AuEH2) are subjected to molecular modification on the basis of rational designs and are combined with site-saturation mutagenesis methods for genes, so that the multiple epoxide hydrolase mutants with the improved enantioselectivity can be obtained. The aspergillus usamii epoxide hydrolase mutants have the advantages that racemic styrene oxide (rac-SO) can be catalyzed by the six mutants AuEH2A250I, AuEH2A250M, AuEH2A250Y, AuEH2A250S, AuEH2A250L and AuEH2A250V with the improved enantioselectivity, and the enantiomeric ratios (E values) of the six mutants can be increased and respectively reach 48, 27.2, 23.9, 21.8, 20.0 and 19.4 from the original 16 as compared with wild type mutants.

Description

technical field [0001] The invention relates to an aspergillus usami epoxide hydrolase mutant with improved enantioselectivity, which belongs to the technical field of enzyme engineering and biocatalysis. Background technique [0002] Chiral epoxides and vicinal diols are important intermediates for the synthesis of various active substances in industries such as chiral drugs, agriculture, spices and fine chemicals, and have broad application prospects and market demands. Photoactive chiral compounds have unique properties different from racemates, and their differences in metabolic pathways, metabolic rates, pharmacology and toxicity in vivo make them have new and special uses in the chemical and life science industries . In the 1960s, the "reaction stop event" that shocked the world has fully demonstrated the necessity of obtaining optically pure compounds. The traditional chemical splitting of epoxides often requires heavy metals and toxic substances as catalysts, which...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N1/21C12P41/00C12P17/02C12R1/19
CPCC12N9/14C12P17/02C12P41/001C12Y303/02003
Inventor 邬敏辰胡蝶王瑞叶慧华李剑芳
Owner JIANGNAN UNIV
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