Enone reductases, methods for producing same, and methods...

Details Enone reductases, methods for producing same, and methods... Enone reductases, methods for producing same, and methods...

2002-02-21

2004-08-24

Stucker, Jeffrey (Department: 1648)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C435S183000

Reexamination Certificate

active

06780976

ABSTRACT:

TECHNICAL FIELD
The present invention relates to novel enone reductases which are useful for reducing an &agr;,&bgr;-unsaturated bond of an &agr;,&bgr;-unsaturated ketone (enone), and polynucleotides encoding such reductases, as well as methods for producing the reductases and methods for selectively reducing a carbon-carbon double bond of an &agr;,&bgr;-unsaturated ketone using the reductases or a polypeptide having homology with the enzyme.
BACKGROUND
Ketones are compounds that are widely used as raw materials in the synthesis of organic compounds. In addition, ketones are also important raw materials for the production of optically active alcohols and optically active amines that are optically active intermediates important in the synthesis of pharmaceuticals. For example, &agr;,&bgr;-unsaturated ketones obtainable by the condensation reaction of aldehydes and ketones are useful as precursors for these ketones.
For example, 3-methyl-3-penten-2-one can be readily prepared by the condensation of acetaldehyde and 2-butane (J. Amer. Chem. Soc., 81:1117-1119, 1959).
Various ketones can be obtained by selectively reducing the &agr;,&bgr;-unsaturated bonds of &agr;,&bgr;-unsaturated carbonyl compounds. Hydrogenation reactions using Ni catalyst or Pd-C catalyst (“Catalytic Hydrogenation Reaction” p135, Tokyo Kagaku Dojin (1987)) are methods known in the art for selectively reducing the &agr;,&bgr;-unsaturated bonds alone, without reducing any carbonyl groups. However, these methods have the following problems to be solved: (1) carbonyl groups may be also reduced by continuing the reaction; (2) metals, which have adverse effects on the environment, are used as the catalysts; and (3) high-pressure hydrogen gas is required for the reaction. Importantly, the reduction of carbonyl groups leads to decrease of the ketone yield.
On the other hand, methods using organisms as follows are reported as methods for selectively reducing carbon-carbon double bonds of &agr;,&bgr;unsaturated ketones using biological reactions:
plant cells (J. Nat. Prod. 56:1406-1409, 1993);
baker's yeast (Tetrahedron Lett. 52:5197-5200, 1978; Bull. Chem. Soc. Jpn. 64:3473-3475, 1991; Tetrahedron Asym. 6:2143-2144, 1995; etc.); and
fungus (J. Org. Chem. 47:792-798, 1982).
However, these biological methods have their own problems such as: (1) carbonyl groups are also reduced; (2) low reactivity; and (3) cell preparation on a large-scale is difficult. Further, various types of enone reductases derived from these organisms have been reported. However, genes encoding these reductases remain to be cloned, and it is therefore hard to conveniently prepare these enzymes on a large scale.
In addition to the above-mentioned reductases of the &agr;,&bgr;-unsaturated carbonyl compounds, such reductases as follows have been reported. These reductases are not suitable for industrial applications because either the substrate specificity of these reductases remains to be clarified or the selectivity for the &agr;,&bgr;-unsaturated bond is low.
Clostridium tyrobutyricum
-derived 2-enoate reductase (E.C.1.3.1.31) (J. Biotechnol. 6:13-29, 1987);
Clostridium kluyveri
-derived acryloyl-CoA reductase (Biol. Chem. Hoppe-Seyler 366:953-961, 1985);
Enone reductase YER-2 purified from baker's yeast (Kawai et al. ((Kyoto University), The 4
th
Biocatalyst symposium, Abstract p58 (2001));
Enone reductases purified from a baker's yeast EI and EII (Eur. J. Biochem. 255:271-278, 1998);
Enone reductase (verbenone reductase; also referred to as p90) derived from tobacco (
Nicotiana tabacum
) cells (J. Chem. Soc., Chem. Commun. 1426-1427, 1993; Chem. Lett. 850-851, 2000);
Carvone reductase (also referred to as enone reductase-I), which is an enone reductase derived from tobacco (
Nicotiana tabacum
) cells (Phytochemistry 31:2599-2603, 1992):
Enone reductase-II, p44, and p74, which are enone reductases derived from tobacco (
Nicotiana tabacum
) cells;
Enone reductases purified from
Euglena gracilis
and
Astasia longa
, which are plant species (Phytochemistry 49, 49-53 (1998)); and
Enone reductase purified from rat liver (Arch. Biochem. Biophys. 282:183-187, 1990).
SUMMARY
The object of the present invention is to provide novel enone reductases, which have an enzyme activity to selectively reduce the &agr;,&bgr;-unsaturated bonds of &agr;,&bgr;-unsaturated ketones to produce &agr;,&bgr;-saturated ketones, and genes encoding the reductases. Another object of the present invention is to provide methods for selectively reducing the carbon-carbon double bonds of &agr;,&bgr;-unsaturated ketones using the reductases and organisms producing the reductases.
The present inventors screened enzymes producing 2-butanone from methyl vinyl ketone and found that
Kluyveromyces lactis
has the activity of interest. Then, they purified the enzyme having the activity of interest from fungal cells of
Kluyveromyces lactis
, and revealed the properties thereof. They confirmed that the enzyme selectively reduced the &agr;,&bgr;-unsaturated bonds of &agr;,&bgr;-unsaturated ketones in a &bgr;-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner, and that the enzyme has substantially no activity to reduce ketones. Further, the present inventors cloned a gene encoding the enzyme, clarified the structure thereof, and verified that the gene was novel. In addition, they overexpressed the gene in a heterologous organism to obtain a transformed strain having higher selectivity and higher activity at the same time to reduce the &agr;,&bgr;-unsaturated bonds of &agr;,&bgr;-unsaturated ketones in a NADPH-dependent manner. Furthermore, they found that selective reduction of the carbon-carbon double bonds of &agr;,&bgr;-unsaturated ketones can be achieved by the enzyme, homologues thereof, cells producing them, and so on, and thus, completed the present invention. Hereinafter, &bgr;-nicotinamide adenine dinucleotide phosphate is referred to as NADP; &bgr;-nicotinamide adenine dinucleotide as NAD; and the reduced forms thereof as NADPH and NADH, respectively.
More specifically, the present invention relates to the following enone reductases, polynucleotides encoding the reductases, methods for producing the reductases, and methods for selectively reducing carbon-carbon double bonds of &agr;,&bgr;-unsaturated ketones using the reductases or polypeptides having homology to such reductases.
[1] An enone reductase having the following physicochemical properties:
(A) Action:
The enzyme reduces the carbon-carbon double bonds of the &agr;,&bgr;-unsaturated ketones, using NADPH as an electron donor, to produce the corresponding saturated hydrocarbon;
(B) Substrate specificity:
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
(1) the enzyme reduces the carbon-carbon double bonds of the &agr;,&bgr;-unsaturated ketones but has substantially no activity to reduce ketones;
(2) the enzyme exhibits a significantly higher activity with NADPH than with NADH as the electron donor;
(3) the enzyme does not substantially act on substrates, wherein both substituents at the &bgr; carbon relative to the ketone are not hydrogen; and
(4) the enzyme does not substantially act on substrates, wherein the carbon-carbon double bond is present in the cyclic structure; and
(C) Optimal pH:
pH 6.5-7.0;
[2] The enone reductase of [1], wherein the reductase further has the following physicochemical properties:
(D) Optimum temperature:
37-45° C.
(E) Molecular weight:
The molecular weight of the reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration is about 43,000 and about 42,000, respectively;
[3] The novel enone reductase of [1], which is derived from the genus Kluyveromyces;
[4] A method for obtaining the enone reductase of [1], comprising the step of culturing a micr

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