(R)-2-octanol dehydrogenase, process for producing enzyme, DNA encoding enzyme and process for producing alcohol by using same

A dehydrogenase, octanol technology, applied in biochemical equipment and methods, botanical equipment and methods, applications, etc., can solve problems such as unfavorable industrial applications, chemically unstable NADPH, etc.

Inactive Publication Date: 2002-08-28
DAICEL CHEM IND LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the synthesis of (R)-propoxybenzene derivatives using this enzyme requires the addition or regeneration of expensive and chemically unstable NADPH, which is unfavorable in industrial applications

Method used

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  • (R)-2-octanol dehydrogenase, process for producing enzyme, DNA encoding enzyme and process for producing alcohol by using same
  • (R)-2-octanol dehydrogenase, process for producing enzyme, DNA encoding enzyme and process for producing alcohol by using same
  • (R)-2-octanol dehydrogenase, process for producing enzyme, DNA encoding enzyme and process for producing alcohol by using same

Examples

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Embodiment 1

[0194] The present invention is illustrated in detail with reference to the following examples, but is not limited thereto. Example 1: Screening of (R)-2-octanol dehydrogenase

[0195] Yeast (R)-2-octanol dehydrogenase is screened by activity staining method after electrophoresis. The substrate is (R)- or (S)-2-octanol substrate. The reaction mixture includes NAD+, phenazine sulfate Dimethyl ester (PMS), nitro blue tetrazolium (NBT). The composition of the reaction mixture is as follows:

[0196] 5mM (R)- or (S)-2-octanol

[0197] 1.3mM NAD+

[0198] 0.13mM PMS

[0199] 0.48m MNBT

[0200] As a result, enzymes that were not stained when the substrate was (S)-2-octanol but preferentially stained when the substrate was (R)-2-octanol were found in the following yeast strains. These enzymes with (R)-2-octanol dehydrogenase activity also have the activity of producing ethyl (S)-4-chloro-3-hydroxybutyrate by reduction of ethyl 4-chloroacetoacetate.

[0201] ●Pichia finlandica...

Embodiment 2

[0204] Ogataea wickerhamii: IFO 1706 Example 2: Culture conditions for production of (R)-2-octanol dehydrogenase

[0205] The strain producing (R)-2-octanol dehydrogenase in Example 1 was cultured in a carbon source where 2% glucose was changed to 2% glycerol or 1% methanol. It turned out that Pichia finlandica could induce this enzyme in all cultures. The enzyme was strongly induced by Candida utilis when methanol was used as the carbon source. Example 3: Purification of (R)-2-octanol dehydrogenase from Pichia finlandica

Embodiment 3

[0206] Pichia finlandica DSM 70280 strain was cultured in 4.8L medium A, and microbial cells were prepared by centrifugation. The obtained microbial cells were suspended in 100 mM Tris-HCl buffer (pH 8.0), 10% glycerol, 1 mM ethylenediaminetetraacetic acid 2Na (EDTA Na), 0.02% 2-mercaptoethanol, and 2 mM benzenemethanesulfonyl fluoride ( PMSF), homogenized with a glass bead stirrer (Biospec). Then, the microbial cell debris is removed by centrifugation to obtain a cell-free extract. Protamine sulfate was added, and the nucleic acid was removed by centrifugation to obtain a supernatant. Ammonium sulfate was added to the supernatant to give a precipitated fraction at 40-75% saturation. A standard buffer solution containing 30% saturated ammonium sulfate was added to the precipitated fraction to obtain an enzyme solution containing 40% (final concentration) saturated ammonium sulfate. The enzyme solution was centrifuged to obtain a supernatant. The supernatant was applied to ...

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Abstract

(R)-2-octanol dehydrogenase that catalyzes oxidation/reduction reactions using NAD+(NADH) as a coenzyme. The enzyme can be obtained from microorganisms of the genus Pichia, Candida, Ogataea and the like. Using this (R)-2-octanol dehydrogenase to reduce ketones can produce alcohols, especially (S)-4-halo-3-hydroxybutyrate and (R)-propoxybenzene derivatives. Moreover, the (R)-2-octanol dehydrogenase has excellent activity and stereoselectivity.

Description

technical field [0001] The present invention relates to novel (R)-2-octanol dehydrogenases useful for the production of alcohols, ketones, especially optically active alcohols such as (S)-4-halo-3-hydroxybutyric acid Esters and (R)-propoxybenzene derivatives; DNAs encoding the enzyme; methods for the production of the enzyme; production of alcohols, ketones, especially optically active alcohols such as (S)-4- Methods of Halo-3-Hydroxybutyrate and (R)-Propoxybenzene Derivatives. technical background [0002] The compound (S)-4-halo-3-hydroxybutyrate is used as an intermediate for the synthesis of HMG-CoA reductase inhibitors, such as D-carnitine and the like. Such compounds can be used in the synthesis of drugs and pesticides. In particular how to obtain (synthesis or decomposition) the optically pure enantiomers of (S)-4-halo-3-hydroxybutyrates is an industrially important problem. Nowadays, methods of asymmetric synthesis, crystallization, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12P7/22
CPCC12N9/0006C12P7/22
Inventor 工藤真丈山本浩明
Owner DAICEL CHEM IND LTD
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