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Halohydrin dehalogenase and its use in synthesis of statin drug intermediate

A halohydrin dehalogenase, halohydrin technology, applied in the field of bioengineering, can solve the problems of inappropriate reaction conditions, high enzyme dosage, long reaction steps and the like

Active Publication Date: 2016-05-11
ABIOCHEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the shortcomings of the prior art, such as excessive enzyme dosage, inappropriate reaction conditions, low reaction conversion rate, and long reaction steps, the present invention provides an environmentally friendly halide with high catalytic activity, high reaction yield, and Alcohol dehalogenase, and using the halohydrin dehalogenase to substitute and synthesize 6-substituted-3,5-dihydroxyhexanoic acid ester derivatives, and an enzyme-chemical synthesis method for further synthesizing statin drug intermediates

Method used

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  • Halohydrin dehalogenase and its use in synthesis of statin drug intermediate
  • Halohydrin dehalogenase and its use in synthesis of statin drug intermediate
  • Halohydrin dehalogenase and its use in synthesis of statin drug intermediate

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: the enzyme activity assay of dehalogenase

[0073] Activity of halohydrin dehalogenase: The catalytic efficiency of the enzyme is evaluated by measuring the concentration of halide ions in a reaction system containing 50mM Tris-SO4Buffer and a certain concentration of substrate.

[0074] Reagent I: 0.25MNH 4 Fe(SO 4 ) 2 Soluble in 9MHNO 3 ; Dilute with 8 times the volume of triple distilled water.

[0075] Reagent II: a saturated solution of Hg(SCN) dissolved in absolute ethanol.

[0076] (1) The substrate is 2-bromoethanol, and potassium bromide or sodium bromide is used as a standard curve; its absorbance at 460 nm is measured with an ultraviolet spectrophotometer, and water is used as a blank control.

[0077] (2) Another approach is to use chlorinated substances as substrates, and use potassium chloride or sodium chloride as a standard curve;

Embodiment 2

[0078] Embodiment 2: screening dehalogenase

[0079] Soil sample DNA (ChromaSpinTE-1000, ClontechLaboratories, Inc., USA) was collected, partially digested with Sau3AI, and 2-8kb fragments were collected by electrophoresis, recovered and ligated into the BamHI site of pUC19 to obtain a plasmid library. Transform the library into E.coliDH10b and smear it on an LB plate containing 100ug / mL ampicillin, select positive clones and transfer them to a 96 deep-well plate with 500uLLB (100ug / mL ampicillin), culture at 37°C for 4 hours, then add 1mMIPTG Induction, continue culturing overnight at 30°C; then take 50uL of each deep-well culture into a new 96-well plate containing 50mM sodium phosphate buffer (pH7.5), freeze and thaw repeatedly at -80°C to lyse the bacteria; add 2mM 6-chloro- tert-butyl 3,5-dicarbonylhexanoate and reagent I and reagent II in Example 1 were incubated at 30°C for 4 hours, and the absorbance was measured at 460nm, and the deep-well culture corresponding to the...

Embodiment 3

[0080] Embodiment 3: Construction and expression of dehalogenase recombinant bacteria

[0081]A primer pair P1 (nucleotide sequence: SeqID NO: 2) and P2 (nucleotide sequence: SeqID NO: 3) was synthesized. P1 and P2 were used to clone the full-length halohydrin dehalogenase gene.

[0082] The PCR system is as follows: 10×KOD-PlusPCR buffer 2μL, 25mMMgSO4 1.2μL, 2mMdNTP 2μL, KOD-PlusPCR high-fidelity enzyme 0.3μL, DNA template 0.5μL (including DNA template 0.1μg), ddH2O 13μL, 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 ...

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Abstract

The invention discloses a halohydrin dehalogenase with high catalytic activity, a high reaction yield and good environmental friendliness and an enzyme chemical synthesis method for replacement synthesis of a 6-substituted-3, 5-dihydroxyhexanoate derivative through the halohydrin dehalogenase and further synthesis of a statin drug intermediate. The invention also provides a nucleic acid sequence for coding the halohydrin dehalogenase, a recombinant expression vector containing the nucleic acid sequence, a recombinant expression transformant, a preparation method of the halohydrin dehalogenase and a use of the halohydrin dehalogenase in a catalytic replacement reaction.

Description

technical field [0001] The present invention relates to the field of bioengineering, in particular to a halohydrin dehalogenase, 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 a method for preparing the recombinant enzyme in catalytic proximity -Applications of haloalcohol compounds in substitution reactions. Background technique [0002] Statins are hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors that block intracellular hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase by competitively inhibiting endogenous cholesterol synthesis rate-limiting enzyme (HMG-CoA) reductase The acid metabolism pathway reduces intracellular cholesterol synthesis, thereby feedback stimulating the number and activity of low-density lipoprotein receptors on the surface of cell membranes (mainly liver cells), increa...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12N15/70C12N1/21C12P13/00C12P7/62
Inventor 罗煜丁时澄瞿旭东王海涛李辉
Owner ABIOCHEM BIOTECH CO LTD
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