Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2001-10-16
2004-03-16
Achutamurthy, P. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S440000, C536S023200
Reexamination Certificate
active
06706507
ABSTRACT:
TECHNICAL FIELD
The present invention relates to novel (R)-2-octanol dehydrogenases useful for producing alcohols, ketones, particularly for producing optically active alcohols such as (S)-4-halo-3-hydroxybutyric acid esters and (R)-propoxybenzene derivatives, DNAs encoding the enzyme, methods for producing the enzymes, and methods for producing alcohols, ketones, particularly for producing optically active alcohols such as (S)-4-halo-3-hydroxybutyric acid esters and (R)-propoxybenzene derivatives using the enzymes.
BACKGROUND
(S)-4-halo-3-hydroxybutyric acid esters are compounds used as intermediates in synthesizing HMG-CoA reductase inhibitors, D-camitine, etc. These compounds are useful for syntheses of medicines and pesticides. Especially, how to get (to synthesize or separate) optically pure enantiomers of (S)-4-halo-3-hydroxybutyric acid esters is industrially important problem. So far, asymmetric synthesis, crystallization, and asymmetric reduction method using microorganisms such as baker's yeast (Unexamined Published Japanese Patent Application (JP-A) Sho 61-146191, JP-A Hei 6-209782, and such) are known as methods for producing (S)-4-halo-3-hydroxybutyric acid esters. However, these known methods are inappropriate for industrial use because of the problems such as low optical purities of products, low yield, etc.
In addition, enzymes that reduce 4-haloacetoacetic acid esters to (S)-4-halo-3-hydroxybutyric acid estersare also being searched. For example, enzymes indicated below are known. The methods for synthesizing (S)4-halo-3-hydroxybutyric acid esters using these enzymes are reported. These enzymes are, however, reductases that use reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme. Therefore, synthesizing (S)-4-halo-3-hydroxybutyric acid esters using these enzymes requires addition and regeneration of NADPH, which is expensive and chemically unstable, and is industrially disadvantageous.
Some reductases derived from baker's yeast (D-enzyme-1, D-enzyme-2, J. Am. Chem. Soc., 107:2993-2994, 1985)
Aldehyde reductase 2 derived from
Sporobolomyces salmonicolor
(Appl. Environ. Microbiol., 65:5207-5211, 1999)
Keto pantothenic acid ester reductase derived from
Candida macedoniensis
(Arch. Biochem. Biophys., 294:469-474, 1992)
4-Chloroacetoacetic acid ethyl ester reductase derived from
Geotrichum candidum
(Enzyme Microb. Technol. 14, 731-738, 1992)
Carbonyl reductase derived from
Candida magnoliae
(WO 98/35025)
Carbonyl reductase derived from
Kluyveromyces lactis
(JP-A Hei 11-187869)
&bgr;-Ketoacyl-acyl carrier protein reductase as one of fatty acid synthases type II (JP-A 2000-189170)
Although 3&agr;-hydroxysteroid dehydrogenase (JP-A Hei 1-277494), glycerol dehydrogenase (Tetrahedron Lett. 29, 2453-2454, 1988), and alcohol dehydrogenase derived from Pseudomonas sp. PED (J. Org. Chem., 57:1526-1532, 1992) are known as reductases using reduced form of nicotinamide adenine dinucleotide (NADH) as a electron donor, these enzymes are industrially disadvantageous because the activity of reaction for synthesizing (S)-4-halo-3-hydroxybutyric acid esters is low.
As indicated above, known methods for producing (S)-4-halo-3-hydroxybutyric acid esters using microorganisms and enzymes were not satisfactory in some respects such as optical purities, yields, activities, etc. These problems have made known methods difficult for industrial use.
On the other hand, (R)-propoxybenzene derivatives (JP-A Hei 02-732) are useful compounds as intermediates in synthesizing medicines, especially, optically active substances of ofloxacin ((S)-(−)-9-fluoro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzooxazine-6-carboxylic acid, JP-A Sho 62-252790), which is synthetic antibacterial drugs. How to get (to synthesize or separate) optically pure enantiomers of these compounds is industrially important problem.
Asymmetric acylation of racemates of propoxybenzene derivatives using lipase and esterase (JP-A Hei 03-183489) is known as a method for producing (R)-propoxybenzene derivatives. In this method, a process to separate remaining raw materials and acylated products after acylation of (R) form and a process to deacylate the acylated products are required. Therefore, this known method is inappropriate for industrial use because these processes are complicated.
The method for asymmetric reduction of acetonyloxybenzene derivatives using microorganisms has been also reported. However, this known method is inappropriate for industrial use because optical purities of (R)-propoxybenzene derivatives produced is as low as 84 to 98% (JP-A Hei 03-183489) or 8.8 to 88.4% (JP-A Hei 05-68577) and because the concentration of substrate is also as low as 0.1 to 0.5%. As the method in which high optical purities can be obtained by asymmetric reduction, the method using carbonyl reductase produced by
Candida magnoliae
(JP-A 2000-175693) was reported to synthesize (R)-propoxybenzene derivatives whose optical purities are 99% or more. However, this carbonyl reductase uses NADPH as a coenzyme. Therefore, synthesizing (R)-propoxybenzene derivatives using this enzyme requires addition and regeneration of NADPH, which is expensive and chemically unstable, and is industrially disadvantageous.
SUMMARY
An objective of the present invention is to provide novel enzymes that can reduce 4-haloacetoacetic acid esters using NADH as a coenzyme and produce (S)-4-halo-3-hydroxybutyric acid esters having high optical purities. Furthermore, an objective of the present invention is to provide methods for producing, using the enzyme, (S)-4-halo-3-hydroxybutyric acid esters having high optical purities.
In addition, an objective of the present invention is to provide novel enzymes that can produce optically highly pure (R)-propoxybenzene derivatives, which are useful as intermediates in synthesizing antibacterial drugs, using NADH as a coenzyme. Furthermore, an objective of the present invention is to provide methods for producing, using the enzyme, (R)-propoxybenzene derivatives that have high optical purities.
The present inventors thought that alcohol dehydrogenase that can use NADH as an electron donor was useful for industrial use. NADH is cheaper and chemically more stable than NADPH. To discover enzymes that can effectively produce optically active (S)-4-halo-3-hydroxybutyric acid esters, the present inventors screened for alcohol dehydrogenase which has high activity on (R)-2-octanol, which has the same configuration as that of (S)-4-halo-3-hydroxybutyric acid esters and which has long chain as long as that of 4-haloacetoacetic acid esters.
Previous findings reported the enzymes derived from
Comamonas terrigena
, Pichia sp. NRRL-Y-11328, and pseudomonas sp. SPD6 as secondary alcohol dehydrogenases that can oxidize (R)-2-octanol stereoselectively and have activities to produce 2-octanone. However, no report has been made that these enzymes can reduce 4-haloacetoacetic acid esters and produce (S)-4-halo-3-hydroxybutyne acid esters. Activities to produce (S)-4-halo-3-hydroxybutyric acid esters by reducing 4-haloacetoacetic acid esters whose carbonyl group is bound to bulky side chains are expected to be low because activities of these enzymes for (R)-2-octanol are not significantly higher than activities for secondary alcohol like 2-propanol, which has short side chains.
Therefore, the present inventors screened widely for microorganisms that possess enzymes having ability to oxidize (R)-2-octanol preferentially. As a result, they have discovered that the microorganisms belonging to the genera below possess enzymes having ability to oxidize (R)-2-octanol preferentially:
Genus Pichia
Genus Candida
Genus Ogataea
Specifically, microorganisms below are found to possess enzymes having ability to oxidize (R)-2-octanol preferentially.
Pichia finlandica
Pichia jadinii
Candida utilis
Ogataea wickerhamii
Moreover, the present inventors cultivated these microorganisms and purified enzymes that can oxidize (R)-2-octanol from the m
Kudoh Masatake
Yamamoto Hiroaki
Achutamurthy P.
Daicel Chemical Industries Ltd.
Pak Yong D.
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