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Biological preparation method of (R)-4-cyano-hydroxybutanoate

A technology of ethyl hydroxybutyrate and biological preparation, which is applied in the field of biopharmaceuticals and green chemistry, can solve the problems of high enzyme dosage, high enzyme feeding amount, and inappropriate reaction conditions, etc., and achieve reduced enzyme dosage, improved purity and yield , the effect of reducing production costs

Active Publication Date: 2013-04-03
ENZYMEWORKS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Afterwards, the domestic patent CN 102168117A improved the process and realized the recovery and utilization of sodium cyanide, but the enzyme dosage was as high as 8.0% (w / w) of the substrate, and the crude yield was only 83.8%.
[0005] Based on the above literature retrieval information, we found that in the existing process, there are defects such as excessive enzyme dosage and improper reaction conditions.

Method used

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  • Biological preparation method of (R)-4-cyano-hydroxybutanoate

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Experimental program
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Embodiment 1

[0024] Inoculate a single colony from a glycerol tube or transformation plate into 4 mL liquid LB medium containing ampicillin resistance and activate overnight (37°C, 200rpm). Transfer 100mL of liquid LB medium containing ampicillin resistance from the overnight culture at 1 / 100 (v / v) inoculum, culture with shaking at 37°C and 200rpm until the OD600 value reaches 0.6-0.8, add IPTG and continue to culture at 30°C overnight. Cells were collected by centrifugation, and suspended in 10 mL of phosphate buffer (2 mM, pH 7.0). The cell suspension was sonicated in an ice bath for 10 minutes. Centrifuge and pre-freeze the supernatant overnight. Freeze-dried for 24h-48h.

Embodiment 2

[0026] In a 50mL three-neck flask, add 23mL of deionized water and 7g of substrate in sequence; after stirring evenly, slowly drop into 5mL of 30% NaCN solution (20% sulfuric acid solution is used to maintain the pH of the system at 7.2 to 7.3 during the dropping process), and the halogenated alcohols are removed. Halogenase powder 35mg, at 50°C, pH 7.0 (30%NaCN solution titration), 800rpm magnetic stirring conditions, react for 24h, GC detection conversion rate> 98%, add sulfuric acid to adjust the pH to 2-3, add an equal volume of acetic acid Ethyl ester, diatomaceous earth filter, collect the organic phase, add an equal volume of ethyl acetate to the aqueous phase to extract twice, combine the organic phase, and rotary evaporate to obtain 6.3g of the product, with a purity of >98% and an optical purity of >99%.

Embodiment 3

[0028] Into the reactor, add 2.3L of deionized water and 700g of substrate in sequence; after stirring evenly, slowly drop into 0.5L of 30% NaCN solution (20% sulfuric acid solution is used to maintain the pH of the system at 7.2~7.3 during the dropping process), halogenated alcohol Dehalogenase powder 3.5g, at 50°C, pH 7.0 (30%NaCN solution titration), under the condition of mechanical stirring, react for 24h, GC detection conversion rate > 98%, add sulfuric acid to adjust the pH to 2-3, add an equal volume Ethyl acetate, filter with diatomaceous earth, collect the organic phase, add an equal volume of ethyl acetate to the water phase to extract twice, combine the organic phases, pass nitrogen gas through the lye to remove HCN gas, and rotary evaporate to obtain about 630g of the product, with a purity > 98%, optical purity >99%.

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Abstract

The invention relates to a biological preparation method of (R)-4-cyano-hydroxybutanoate. According to the biological preparation method, (S)-4-chlorine-3-hydroxybutanoate is used as a substrate; under the action of a biocatalyst, the substrate reacts with sodium cyanide to generate a target product (R)-4-cyano-hydroxybutanoate; the biocatalyst is recombination halohydrin dehalogenase; and the reaction is carried out in a water phase with the pH of 6 to 8. According to the invention, by adopting the recombination halohydrin dehalogenase, the concentration of the substrate is improved and the enzyme dosage is reduced, so that production cost is reduced. Moreover, by optimizing the process, both purity and yield of the product are improved.

Description

technical field [0001] The invention belongs to the fields of biopharmaceuticals and green chemistry, in particular to a biological preparation method of (R)-4-cyano-3-hydroxybutyrate ethyl Background technique [0002] Atorvastatin (trade name Lipitor, Lipitor) is the world's first drug with annual sales of more than 10 billion US dollars. It lowers blood cholesterol levels by inhibiting hydroxymethylglutaryl-CoA reductase, the rate-limiting step in cholesterol synthesis in the liver. [0003] The key step of present technique is to utilize (S)-4-chloro-3-hydroxybutyric acid ethyl ester (A4) one-step synthesis (R)-4-cyano-3-hydroxybutyric acid ethyl ester (A5) (Angew. Chem. Int. Ed. 2005, 44:362-365). [0004] In this step reaction, because the biological method avoids the shortcomings of too many by-products formed under alkaline conditions in the chemical method, and the shortcomings of difficult separation and purification, the synthesis of A5 from A4 using halohydrin ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/00
Inventor 鞠鑫尹将来张超
Owner ENZYMEWORKS
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