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(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

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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

This invention provides (R)-2-octanol dehydrogenase that catalyzes oxidation-reduction reaction using NAD<+> (NADH) as a coenzyme and the genes that encodes them. The enzymes of this invention can be obtained from microorganisms such as the genera Pichia, Candida, and Ogataea, and so on. It is possible to produce alcohols, in particular, alcohols such as (S)-4-halo-3-hydroxybutyric acid esters and (R)-propoxybenzene derivatives by reducing ketones with this (R)-2-octanol dehydrogenase. Moreover, the (R)-2-octanol dehydrogenase of this invention is excellent in 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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