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Enol reductase mutant and application thereof in preparation of (R)-citronellal

A reductase and mutant technology, applied in the field of enol reductase mutants and its application in the preparation of (R)-citronellal, to achieve high stereoselectivity

Active Publication Date: 2020-07-28
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the molecular transformation of the 296th serine of OYE3 to phenylalanine and the 116th tryptophan mutation to glycine, nor the use of OYE3-Mut to catalyze the asymmetric reduction of (E)-citral or Synthesis of (R)-citronellal from (E / Z)-citral

Method used

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  • Enol reductase mutant and application thereof in preparation of (R)-citronellal
  • Enol reductase mutant and application thereof in preparation of (R)-citronellal
  • Enol reductase mutant and application thereof in preparation of (R)-citronellal

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Construction of genetically engineered strain E.coli BL21(DE3) / pET28b-OYE3

[0026] The enol reductase OYE3 encoding gene derived from Saccharomyces cerevisiae (nucleotide sequence is shown in SEQ ID NO. 1) was codon optimized, and the nucleotide sequence of the codon-optimized OYE3 gene was shown in SEQ ID NO. 2 As shown, the amino acid sequence corresponding to the enol reductase OYE3 is shown in SEQ ID NO. 3, synthesized by Hangzhou Kinco Biotechnology Co., Ltd. The nucleotide sequence of the codon-optimized OYE3 gene (SEQ ID NO.2) was artificially synthesized and inserted between the Nco I and Xho I of pET28b to obtain the recombinant plasmid pET28b-OYE3, see the electrophoresis diagram figure 2 Shown.

[0027] Take out 5μL of synthetic recombinant expression plasmid pET28b-OYE3 and add it to 50μL E.coli BL21(DE3) competent, flick the tube wall to mix, and place on ice for 30min. Heat shock in 42℃ water bath for 45s, and immediately place on ice for 2min. A...

Embodiment 2

[0029] Example 2: Construction of recombinant expression plasmid pET28b-OYE3-Mut and genetically engineered strain E.coli BL21(DE3) / pET28b-OYE3-Mut

[0030] 1. Recombinant expression plasmid pET28b-OYE3-Mut

[0031] Using the plasmid pET28b-OYE3 prepared in Example 1 as a template, primers F1 and R1 were designed, and the whole plasmid was cloned by inverse PCR technology and transformed into E. coli BL21 (DE3). The plasmid was extracted and sequenced, and the sequencing results were analyzed by software. The sequence contained an open reading frame of 1200 bp, and the 296th amino acid was successfully changed from serine to phenylalanine. Then use the mutant plasmid as a template to design F2 and R2, and clone the whole plasmid by inverse PCR to obtain a mutant plasmid in which the 296th serine is mutated to phenylalanine and the 116th tryptophan is mutated to glycine. The mutant plasmid was transformed into E.coli BL21(DE3), the plasmid was extracted and sequenced, and the mutan...

Embodiment 3

[0046] Example 3: Construction of genetically engineered strain E.coli BL21(DE3) / pET28b-GDH

[0047] Recombinant glucose dehydrogenase GDH encoding gene derived from Exiguobacterium (nucleotide sequence shown in SEQ ID NO. 6 and amino acid sequence shown in SEQ ID NO. 7), synthesized by Hangzhou Kinco Biotechnology Co., Ltd. The nucleotide sequence of GDH gene (SEQ ID NO. 6) was artificially synthesized and inserted between Nde I and Xho I of pET28b to obtain the recombinant plasmid pET28b-GDH.

[0048] Take out 5μL of synthetic recombinant expression plasmid pET28b-GDH and add it to 50μL E.coli BL21(DE3) competent, flick the tube wall to mix, and place on ice for 30min. Heat shock in 42℃ water bath for 45s, and immediately place on ice for 2min. Add 1 mL of LB liquid medium to the tube and incubate on a shaker at 37°C for 1 hour. The culture broth was centrifuged at 4500 rpm for 4 min, and 800 μL of supernatant was taken out. Use the remaining culture medium to suspend the bact...

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Abstract

The invention discloses an enol reductase mutant and application thereof in the preparation of (R)-citronellal. Mutant OYE3-Mut is used as a biocatalyst, NADP+ is used as a coenzyme, glucose dehydrogenase and D-glucose drive a coenzyme cycle, and a double-enzyme cascaded catalysis reaction system for asymmetric synthesis of optically pure (R)-citronellal is successfully constructed. OYE3-Mut doesnot use (Z)-Citral as a substrate. In the double-enzyme catalysis system, the asymmetric reduction of 20 mM (E)-Citral and (E / z)-Citral is catalyzed by an OYE3-Mut enzymatic method for 11 h, respectively. Compared with enol reductase OYE3, the (E)-Citral catalyzes a product to increase e.e. value from 63.4% (R) to 99% (R), and (E / z)-Citral catalyzes the product to increase e.e. value from 23.6% (R) to 99% (R).

Description

[0001] (1) Technical field [0002] The invention relates to an enol reductase mutant OYE3-Mut and its application in the asymmetric synthesis of (R)-citronellal by a biological enzymatic method. [0003] (2) Background technology [0004] Citronellal exists in the metabolites of plants, and it can be obtained by distillation of essential oils of plants or direct extraction from solvents, mostly in the form of (R / S)-citronellal mixtures. The single-configuration (R)-citronellal is an important component of plant essential oils, and has a wide range of applications in spice raw materials, food flavors and reducing the bitterness of caffeine. (R)-Citronellal is a key intermediate for the synthesis of L-menthol. In recent years, studies have also found that (R)-citronellal can specifically inhibit breast cancer cells when it has a small effect on body cells. At the same time, (R)-citronellal can also be used as male wax moth sex pheromone ((5R / 11R) dimethyl behene) and gastric acid se...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12P7/24C12R1/19
CPCC12N9/0008C12N15/70C12P7/24
Inventor 应向贤魏冉汪钊
Owner ZHEJIANG UNIV OF TECH
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