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Alcohol dehydrogenase and uses thereof in synthesis of Duloxetine intermediate

An alcohol dehydrogenase and reaction technology, applied in the field of bioengineering, can solve the problems of incomplete reaction, low yield, high cost of raw materials and the like

Active Publication Date: 2016-01-27
ABIOCHEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is, low yield, high cost of raw materials, incomplete reaction, Corresponding to problems such as low selectivity or the addition of expensive coenzymes, an alcohol dehydrogenase with high catalytic activity, strong enantioselectivity, and good substrate tolerance is provided, and the alcohol dehydrogenase is used to catalyze the synthesis of (S)-3-substituted amino-1-(thiophen-2-yl)-1-propanol, and then the enzymatic-chemical synthesis method of further synthesizing duloxetine

Method used

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  • Alcohol dehydrogenase and uses thereof in synthesis of Duloxetine intermediate
  • Alcohol dehydrogenase and uses thereof in synthesis of Duloxetine intermediate
  • Alcohol dehydrogenase and uses thereof in synthesis of Duloxetine intermediate

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Experimental program
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Effect test

Embodiment 1

[0048] Collect soil samples from Zhujia Village, Situan Town, Fengxian District, Shanghai and extract DNA (extraction method refers to ChromaSpinTE-1000, Clontech Laboratories, Inc., USA), partially digest with Sau3AI, collect 2-8kb fragments by electrophoresis, recover and connect to BamHI site of pUC19 to obtain a plasmid library. Transform the library into E.coliDH5α and smear it on an LB plate containing 100 μg / mL ampicillin. Select positive clones and transfer them to a 96-deep-well plate with 500 μg / mL LB (100 μg / mL ampicillin). After culturing at 37°C for 4 hours, add 1 mMIPTG Induction, continue to culture overnight at 30°C; then take 50 μL of deep-well culture each to a new 96-well plate added with 50mM sodium phosphate buffer (pH7.5), freeze and thaw repeatedly at -80°C to lyse the bacteria; add 1mM duloxetine Substrate DKTP, 10mM glucose, 1 unit of glucose dehydrogenase, 0.002% (v / v) phenol red, cultured at 30°C for 4 hours, picked the deep well culture correspondin...

Embodiment 2

[0050] A primer pair P1 (nucleotide sequence: SEQ ID NO: 3) and P2 (nucleotide sequence: SEQ ID NO: 4) was synthesized. Use P1 and P2 to clone the full-length alcohol dehydrogenase gene, the PCR system is as follows: 10×KOD-Plus PCR buffer 2μL, 25mM MgSO 4 1.2 μL, 2 mMdNTP 2 μL, KOD-PlusPCR high-fidelity enzyme 0.3 μL, DNA template 0.5 μL (including 0.1 μg DNA template), ddH 2 O13μL, P1 and P2 each 0.5μL (10mmol / L). PCR amplification steps are: (1) 95°C, pre-denaturation for 3min; (2) 98°C, denaturation for 15s; (3) 58°C annealing for 30s; (4) 72°C extension for 1min; steps (2) to (4) repeated 30 times; (5) Continue extending at 72°C for 10 minutes, then cool to 4°C. The PCR product was purified by agarose gel electrophoresis, and the target band in the 700-800bp range was recovered using an agarose gel DNA recovery kit (see figure 1 ), obtained a complete gene sequence, which was 750bp in length after DNA sequencing.

[0051] After the PCR product was cut and recovered, it ...

Embodiment 3

[0053] Enzyme activity determination: In 2mL reaction solution, add 2mM MDKTP as substrate, 0.1mM NADH as cofactor, then add 20μL crude enzyme solution, measure OD within 2 minutes 340 The rate of decrease is ΔA 340 . Enzyme activity calculation formula: ΔA 340 ×1000 / (6220×20), that is, the specific enzyme activity per mL of lysate.

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Abstract

The invention provides an alcohol dehydrogenase with high catalytic activity, strong enantiotropic selectivity and good substrate tolerance, and provides an enzymatic synthesis method of employing the alcohol dehydrogenase to catalyze and synthesize (s)-3-substituted amido-1-(thiophenyl-2-yl)-1- propanol and furthermore Duloxetine. The invention also provides a nucleic acid sequence for encoding the alcohol dehydrogenase, a recombinant expression vector including the nucleic acid sequence, and a recombinant expression transformant, and a preparation method of the alcohol dehydrogenase, and uses of the alcohol dehydrogenase in catalyzing asymmetric reduction of carbonyl substrates. The invention has advantages that the product concentration is high, the optical purity of the product is high, the reaction condition is mild and environment-friendly, the operation is convenient and is easy to industrialize, and an extra expensive coenzyme NADP<+> / NAD<+> is not necessary.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to an alcohol dehydrogenase, a recombinant expression vector containing a nucleic acid sequence encoding the enzyme, a recombinant expression transformant, an expressed recombinant enzyme and a preparation method of the recombinant enzyme, and the alcohol dehydrogenation Application of enzymes as catalysts in the asymmetric synthesis of duloxetine intermediates. Background technique [0002] Depression, also known as depressive disorder, is the main type of mood disorder with significant and persistent low mood as the main clinical feature. Depression includes unipolar depression (i.e. major depressive disorder and psychotic depression), adjustment disorder, minor depression, seasonal affective disorder (SAD), premenstrual dysphoric disorder (PMDD), postpartum depression, SARS depression, bipolar disorder, and manic depression. The pathogenesis of depression has not been f...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P17/00
Inventor 罗煜丁时澄瞿旭东王海涛
Owner ABIOCHEM BIOTECH CO LTD
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