Microbial production of actinol

Details Microbial production of actinol Microbial production of actinol

1999-08-16

2002-09-03

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Process of utilizing an enzyme or micro-organism to destroy...

Resolution of optical isomers or purification of organic...

C435S148000, C435S170000

Reexamination Certificate

active

06444460

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a process for the microbial production of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (“actinol”) from (6R)-2,2,6-trimethylcyclohexanedione (“levodione”). Actinol is useful for the synthesis of carotenoids, such as zeaxanthin. More particularly, the present invention relates to a process for the microbial production of actinol utilizing a specific microorganism that is capable of selectively asymmetrically reducing the carbonyl group at the C-4 position of levodione.
BACKGROUND OF THE INVENTION
Actinol has previously been prepared by optical resolution of the diastereomeric mixture of actinol. However, this process requires hydrogenation of levodione by metal catalysts and subsequent optical resolution by chemical means with resolving agents, such as, maleic anhydride (T. Ohashi et al., the proceedings of the symposium “Molecular Chirality 1996” held on May 30 and 31, 1996, in Tokyo, Japan, pages 47 to 50, “Practical Syntheses using Biocatalysts”). Accordingly, this process is not economically feasible for industrial purposes.
Processes of enzymatic preparation of actinol from levodione per se are known. For example,
Bacillus thermophilus
is capable of converting racemic dihydrooxoisophorone to 4 isomers of 4-hydroxy-2,2,6-trimethylcyclohexanone, i.e., to the cis-(4R,6S)-, cis-(4S,6R)-, trans-(4R,6R)- and trans-(4S,6S)-isomers. The resulting quantitative ratio of these isomers is 68:25:5:2 (J. Biotechnol., 9(2), 117 -128, 1989). Because the content of the (4R,6R)-isomer, actinol, is only 5% of the total isomers, this process is also not economically feasible for industrial purposes.
SUMMARY OF THE INVENTION
As a result of extensive studies on selective asymmetric reduction of levodione, it has surprisingly been found that actinol can be obtained efficiently from levodione by selective asymmetric reduction using certain microorganisms followed by recovery of the actinol from the reaction mixture. The present invention is based upon this finding.
Accordingly, the present invention provides a process for making actinol by contacting levodione with a microorganism, which is selected from the group consisting of microorganisms of the genera Cellulomonas, Corynebacterium, Planococcus and Arthrobacter and which is capable of selective asymmetric reduction of levodione to actinol, and recovering the resulting actinol from the reaction mixture.
DETAILED DESCRIPTION OF THE INVENTION
Screening was effected using a method known per se. For example, a microorganism is cultivated in a nutrient medium containing saccharides, such as, glucose and sucrose, alcohols, such as, ethanol and glycerol, fatty acids, such as, oleic acid and stearic acid or esters thereof, or oils, such as, rapeseed oil and soybean oil as carbon sources; ammonium sulfate, sodium nitrate, peptone, amino acids, corn steep liquor, bran, yeast extract and the like as nitrogen sources; magnesium sulfate, sodium chloride, calcium carbonate, potassium monohydrogen phosphate, potassium dihydrogen phosphate and the like as inorganic salt sources; and malt extract, meat extract and the like as other nutrient sources by a conventional method to provide cells. The cultivation can be carried out aerobically, normally for a cultivation period of 1 to 7 days at a medium pH of 3 to 9 and a cultivation temperature of 10 to 40° C. After the cultivation, the resulting cells are collected by centrifugation or filtration. The cells thus obtained and levodione are brought (contacted) together in a solvent such as water, potassium phosphate buffer, acetonitrile, ethanol and the like, and a reaction is initiated under appropriate reaction conditions (levodion concentration: 400 to 2000 mg/g dry cells/l, pH range: 4 to 9, temperature range: 20 to 50° C., reaction period: 10 minutes to 80 hours). The reaction mixture is extracted with an organic solvent such as ethyl acetate, n-hexane, toluene, n-butyl acetate and the like. The extracted solution is subjected to an appropriate method, such as, chromatography, to measure the productivity of actinol from levodione.
As a result of the screening, it has been found that microorganisms belonging to the genera Cellulomonas, Corynebacterium, Planococcus and Arthrobacter are capable of the selective asymmetric reduction of levodione. Preferred such microorganisms are Cellulomonas sp. AKU672,
Corynebacterium aquaticum
AKU610,
Corynebacterium aquaticum
AKU611,
Planococcus okeanokoites
AKU152 and
Arthrobacter sulfurous
AKU635. Especially preferred are the first three named microorganisms, of which
Corynebacterium aquaticum
AKU611 is most preferred.
The microorganisms Cellulomonas sp. AKU672,
Corynebacterium aquaticum
AKU610 and
Corynebacterium aquaticum
AKU611 were isolated from soil samples collected at Lake Manahime, Fukui Prefecture, Japan. These micoorganisms were deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan on Aug. 4, 1998 under the Budapest Treaty and have the following designations: Cellulomonas sp. AKU672 (FERM BP-6449)
Corynebacterium aquaticum
AKU610 (FERM BP-6447)
Corynebacterium aquaticum
AKU611 (FERM BP-6448)
Cellulomonas sp. AKU672,
Corynebacterium aquaticum
AKU610, and
Corynebacterium aquaticum
AKU611 were deposited on Aug. 4, 1998 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-Chome, Tsukuba City, Ibaraki Pref., Japan) as FERM BP-6449, FERM BP-6447, FERM BP-6448, respectively under the terms of the Budapest Treaty. All restrictions on public access to these deposits will be irrevocably removed upon the grant of a patent on this application.
These three microorganisms, and also
Planococcus okeanokoites
AKU152 and
Arthrobacter sulfureus
AKU635, are new and represent a further aspect of the present invention.
The above-mentioned strain AKU672 (FERM BP-6449) has the following taxonomical properties:
Typical pleomorphism of strain Cellulomonas sp. AKU672 was found on electron microscopic observation. An old culture of the strain was coccoidal as shown in FIG.
1
. In young cultures, irregular rods were dominant (FIG.
2
). The morphological, physiological and biochemical characteristics of the strain are summarized in Tables I and II.
TABLE I
Morphological and Culture Characteristics of Strain
Cellulomonas sp. AKU672
Form and size
Coryneform
Old culture; coccoid cells, ca. 0.5-0.6 &mgr;m
Fresh culture; irregular rods, 0.5-0.7 &mgr;m by 20 or
more &mgr;m
Motility
Motile with one flagellum
Gram strain
+
Spores
No observation
Nutrient agar plate
Circular, convex, smooth, entire, yellow (2 days)
Nutrient broth
Ring and slight sediment
Gelatin stab
Liquefaction
Litmus milk
Acid formation
Relation to NaCl
Grow up to 5% NaCl
TABLE II
Physiological and Biochemical Characteristics of Strain
Cellulomonas sp. AKU672
Type of cell wall
Ornithine
Type of cell division
Bending
GC content (%)
74.7%
Hydrolysis of gelatin
+
Hydrolysis of starch
+
Production of indole
−
Production of hydrogen
−
sulfide
Reduction of nitrate
+
to nitrite
Utilization of citrate
−
Catalase activity
+
Oxidase activity
−
Urease activity
−
DNase activity
+
Amino peptidase
−
activity
Cellulose attack
−
Voges-Proskauer test
−
Methyl-red test
−
Oxidation-
Fermentation
Fermentation test*
Cleavage of
Acid production but no gas from arabinose,
carbohydrates
arbutin, cellobiose, dextrin, fructose, galactose,
glucose, glycogen, maltose, starch, sucrose,
trehalose and xylose;
No acid production from glycerol, inulin,
lactose, mannitol, mannose, &agr;-
methylglucoside, raffinose, rhamnose,
sorbitol and sorbose
Optimum temperature
37-42° C.
for growth
Optimum pH for
pH 6.0-7.5
growth
Heating at 63° C. for
Survives
30 min. in skimmed
milk
Aerobic or anaerobic
Aerobic
*R. Hugh & E. Leifson, J. Bacteriol. 66, 24(1953)
The strain Cellulomonas sp. AKU672 is gram-positive and aerobic, and can be classified as belonging to the group

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