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Process for producing phorenol

A technology of phorenol and ketoisophorone, which is applied in the field of producing phorenol, and can solve the problems of low optical purity of the product and other problems

Inactive Publication Date: 2005-09-07
DSM IP ASSETS BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But these methods are inefficient for the optical purity of the product and require multiple steps
There is no biological method to directly produce optically active phorenol

Method used

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  • Process for producing phorenol

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Experimental program
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Effect test

Embodiment approach

[0027] Column: ULBON HR-20M (Shinwa, Japan) 0.25mmφ×30m

[0028] Column temperature: 160°C (constant)

[0029] Syringe temperature: 250°C

[0030] Carrier gas: He (about 1ml / min)

[0031] After the reaction, Forsenol in the reaction mixture can be recovered, for example, by extraction with a water-immiscible organic solvent that readily dissolves Forsenol, such as ethyl acetate, n-hexane, toluene, or n-butyl acetate. Finish. Further purification of forsenol can be done by concentrating the extract to directly crystallize forsenol or by a combination of various chromatography (such as thin-layer chromatography, adsorption chromatography, ion-exchange chromatography, gel filtration chromatography or high-performance liquid chromatography). phase chromatography) to achieve.

[0032] The following examples further illustrate the invention.

Embodiment 1

[0033] Example 1: Production of Forsenol using Corynebacterium aquaticus AKU611 (FERM BP-6448)

[0034] Corynebacterium aquaticus AKU611 (FERM BP-6448) was inoculated in 1.0g / L yeast extract, 15.0g / L Bacto-peptone (Difco laboratories, U.S.A), 0.2g / LMgSO 4 ·7H 2 O, 3.0g / LK 2 HPO 4 , 2.0g / L NaCl and 22.4g / L glucose·H 2 O seed medium (100 mL, in a 500 mL flask), and cultivated at 30° C. with rotary shaking for 24 hours. Part (100ml) of the seed culture was inoculated with 8.0g / L yeast extract, 0.2g / L MgSO 4 ·7H 2 O, 0.01g / L MnSO 4 ·4-5H 2 O, 2.0g / L NaCl and 11.1g / L glucose·H 2Production medium for O (3.0 L, in a 5-L scale fermenter; type MJ-5-6, L.E. Marubishi, Japan). Culture was performed at 30°C with agitation at 600 r.p.m. and aeration at 1.0 vvm. The pH was maintained at 7.0 by using ammonium solution. After approximately 9 hours of incubation, glucose supplementation was started at a feed rate of 20 g / hour. After 24 hours from the start of fermentation, each con...

Embodiment 2

[0035] Embodiment 2: Clone L-diketone reductase gene from Aquatic Corynebacterium AKU611 (FERM BP-6448) genomic DNA

[0036] Genome DNA of Corynebacterium aquaticus AKU611 (FERMBP-6448) was prepared using Genome Isolation Kit (BIO101). Using the prepared genomic DNA as a template, a thermal cycler (Perkin elmer 2400, U.S.A.) was used to amplify the full-length coding sequence of the L-diketone reductase gene without redundant flanking regions by PCR amplification. The two synthetic primers used are as follows:

[0037] LV-ORF(+): (5'-GGAGGC GAATTC ATGACCGCAACCAGCTCC-3') (SEQ ID NO: 1)

[0038] (The underlined sequence is the position of the EcoRI site)

[0039] LV-ORF(+): (5'-GGGCTG CTGCAG TCAGTACGCGGCGGA-3') (SEQ ID NO: 2)

[0040] (The underlined sequence is the position of the PstI site)

[0041] The PCR mixture (0.02 ml) contained 5 pmol of each primer, 0.2 mM of each dNTP, and 1 U of LA Taq (Takara Shuzo co. LTD / Kyoto, Japan). The initial template denaturation ste...

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Abstract

The present invention relates to a process for producing (4S)-4-hydroxy-2,6,6-trimethyl-2-cyclohexene-1-one (phorenol) from 2,6,6-trimethyl-2-cyclohexene-1,4-dione (ketoisophorone) comprising contacting ketoisophorone with a microorganism which is capable of producing actinol from levodione or with a cell-free extract thereof, with a recombinant microorganism which is capable of producing actinol from levodione or with a cell-free extract thereof, or with levodione reductase, and isolating the resulting phorenol from the reaction mixture.

Description

technical field [0001] The present invention relates to the production of (4S)-4-hydroxy-2 from 2,6,6-trimethyl-2-cyclohexene-1,4-dione (hereinafter referred to as ketoisophorone) , 6,6-trimethyl-2-cyclohexen-1-one (hereinafter referred to as Phorenol (Phorenol)) method. More specifically, the present invention relates to the production of forsenol from ketoisophorone by specific microorganisms, cell-free extracts thereof, recombinant microorganisms or cell-free extracts of recombinant microorganisms, or levo-diketone reductase method. Background technique [0002] Forsenols are useful chiral building blocks of naturally occurring optically active compounds such as zeaxanthin. So far, some enantioselective methods for producing furenol have been reported [Tanaka et al., Tetrahedron: Asymmetry 6: 1273 (1995); Kiyota et al., Tetrahedron: Asymmetry 10: 3811 (1999)]. But these methods are inefficient with regard to the optical purity of the product and require multiple steps....

Claims

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Application Information

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IPC IPC(8): C12P7/38C12N9/02C12P7/26C12R1/15
CPCC12P7/26C12N9/0004
Inventor 星野达雄田畑和之濑户口丰清水昌
Owner DSM IP ASSETS BV
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