Alcohol dehydrogenase and its coding gene and its application in catalytic synthesis of (r)-phenylethylene glycol

A technology of phenylethylene glycol and alcohol dehydrogenase, applied in the field of enzyme engineering, can solve the problems of unsatisfactory, high cost, low selectivity of chemical synthesis method, etc., and achieve the effects of improving efficiency, promoting efficiency and reducing inhibition

Active Publication Date: 2022-05-20
HEBEI UNIVERSITY
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Problems solved by technology

[0005] The purpose of the present invention is to provide a kind of alcohol dehydrogenase and its coding gene and the application in catalytic synthesis (R)-phenylethylene glycol, to solve the existing chemical synthesis method low selectivity, high cost, discharge of three wastes and Problems with unsatisfactory substrate conversion rate and product optical purity in existing biocatalytic methods

Method used

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  • Alcohol dehydrogenase and its coding gene and its application in catalytic synthesis of (r)-phenylethylene glycol
  • Alcohol dehydrogenase and its coding gene and its application in catalytic synthesis of (r)-phenylethylene glycol
  • Alcohol dehydrogenase and its coding gene and its application in catalytic synthesis of (r)-phenylethylene glycol

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Embodiment 1

[0026] Example 1 Genome Mining of Microbacterium Alcohol Dehydrogenase and Construction of Recombinant Strains

[0027] Use Primer Premier software (Premier5.5, Palo Alto, CA, USA) to carry out primer design according to the oxidoreductase sequence deduced from the genome (GCF_000422405.1) of Microbacterium luticocti DSM 19459 registered in the NCBI database (as shown in Table 1 Show).

[0028] Table 1: Primer sequences

[0029] name Sequence (5'-3') name Sequence (5'-3') F1 ATGACNMGNACNGCNYTNATHACNG F7 ATGACNGGNGGNCARCCNCARTGGA R1 NCKNCCNCKNGCNGCNGCNCKNACN R7 NGCNARYTCNGCDATDATNGGRTGD F2 ATGGCNACNGAYWSNGCNYTNGAYG F8 ATGACNMGNYTNGGNMGNWSNGAYY R2 CCANSWNSWYTTNCKRTARTCYTTN R8 RTCYTGNGCNGCRTCNSWNGCNCKN F3 ATGGCNGGNACNWSNGGNCAYGTNA F9 ATGWSNMGNTTYACNGGNAARGTNG R3 YTGNACNSWCCANCCNCCRTCNSWN R9 RTGNGCNSWRTANCCNCCRTCNACN F4 ATGACNGAYGCNMGNAAYGAYGAYG F10 ATGCCNTAYMGNGTNATHGTNACNG R4 NGCNGGNGCRTCDATNCCD...

Embodiment 2

[0039] The construction of embodiment 2 double enzyme coupling catalytic system

[0040] The recombinant protein FDH from Candida parapsilosis was passed through the purification system ( GE Healthcare), the affinity column was filled with prepacked Ni Sepharose TM (HisTrap HP; GE Healthcare, Piscataway, NJ, USA). Formate dehydrogenase FDH activity was determined by measuring the increase in absorbance at 340 nm. The reaction mixture (0.1 mL) for the enzyme assay included 0.1 M sodium formate, 1 mM NAD+, 0.1 M KHPO 4 buffer (pH 7.5) and the appropriate enzyme. The specific activity of the enzyme is 140U / mg. Purified MLADH and FDH (100 μL each) were added to a potassium phosphate buffer reaction system (1 mL) containing 1.0 g / L 2-HAP, 1.0 g / L sodium formate and 2 mM NAD+. The reaction system was transformed at 37°C and 200r / min for 2h. After the reaction was completed, the reaction solution was centrifuged, and 100 μL of the supernatant was dissolved in 900 μL of methanol ...

Embodiment 3

[0044] The optimization of embodiment 3 two-phase catalytic system

[0045] Firstly, the biphasic catalytic system of dual enzyme reaction was optimized. The reaction mixture (10 mL) consisted of 0.1 M potassium phosphate buffer (pH 7.0) containing 0.5 g / L sodium formate, 2 mM NAD+, two enzymes (MLADH:FDH=1:10) and an organic solvent (phthalic acid Dibutyl ester, ethyl caprate, ethyl laurate, n-heptane, ethyl acetate, n-hexanol, n-hexane or soybean oil), the concentration of 2-HAP is 5g / L, and the ratio of aqueous phase to organic phase is 1:1. The mixture was stirred at 37 °C for 2 h. Sample 100 µL of each organic layer and dissolve in 900 µL methanol. Product content was determined by HPLC. In addition, the biotransformation of 2-HAP by the dual enzymes was also analyzed using different ratios of aqueous and organic phases (1:0, 4:1, 3:1, 2.3:1, 1.5:1 and 1:1).

[0046] Secondly, the optimal initial concentration of the substrate 2-HAP in the catalytic system was explor...

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Abstract

The invention provides a kind of alcohol dehydrogenase and its encoding gene and its catalytic synthesis ( R )-phenylethylene glycol, the amino acid sequence of the alcohol dehydrogenase is shown in sequence 1 in the sequence listing, and its gene sequence is shown in sequence 2 in the sequence listing. alcohol dehydrogenase gene mladh In catalytic synthesis ( R )‑Phenylglycol. Alcohol dehydrogenase MLADH and formate dehydrogenase FDH were used to construct a dual-enzyme coupled catalytic system. In the system, the enzyme activity ratio of alcohol dehydrogenase MLADH and formate dehydrogenase FDH was 1:5-10. The invention constructs a double-enzyme coupling system of alcohol dehydrogenase MLADH and formate dehydrogenase FDH, and realizes the in-situ regeneration of coenzyme NADH. In the present invention, soybean oil is introduced into the system using phosphate buffer salt as the water phase as the organic phase, which reduces the inhibition of the catalytic reaction by the substrate and the product, promotes the efficiency of the catalytic conversion, and the conversion rate reaches 99%. e.e. The value is above 99%.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to an alcohol dehydrogenase and its coding gene and its application in catalytic synthesis of (R)-phenylethylene glycol. Background technique [0002] (R)-Phenylethylene glycol ((R)-PED) is an important chiral model compound, which can be used to synthesize β-lactam antibiotics, (R)-norfluoxetine, (R)- Fluoxetine, (R)-{N-1-[(3-methylmercaptophenyl)ethyl]}-3-phenyl-1-propylamine and other chiral drugs are used to treat mental disorders, metabolic disorders and Hyperparathyroidism and other diseases. In addition, (R)-PED can also be further made into (R)-mandelic acid, which is often used as a side chain modifier of the cephalosporin antibiotic cephalosporin oxybenzyl tetrazole. [0003] The production methods of (R)-PED are mainly divided into chiral resolution and asymmetric synthesis. The chiral resolution method refers to the conversion of the racemate into a single ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/22C12R1/19
CPCC12N9/0006C12N15/70C12P7/22Y02P20/55
Inventor 李玮张红蕾杜洁张超韩孟楠王旭明傅双庆
Owner HEBEI UNIVERSITY
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