Esterase gene and its use

Details Esterase gene and its use Esterase gene and its use

1998-12-23

2002-10-29

Bugaisky, Gabrielle (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

C435S149000, C435S135000, C435S280000

Reexamination Certificate

active

06472189

ABSTRACT:

FIELD OF INVENTION
The present invention relates to an esterase gene and its use.
BACKGROUND OF THE INVENTION
Cyclopentenolones of formula I:
wherein R
1
is hydrogen or methyl, and R
2
is C
1
-C
10
alkyl, C
2
-C
10
alkenyl, C
2
-C
10
alkynyl, C
1
-C
4
haloalkyl, a C
5
-C
9
hydroxy aliphatic hydrocarbon moiety which may be optionally protected on the terminal hydroxyl group thereof, or a C
5
-C
9
fatty acid residue which may be optionally protected on the terminal carboxyl group thereof, are important as the intermediates of drugs, agricultural chemicals or other active products.
These cyclopentenolones are, useful as the important alcohol components in a group of ester compounds, commonly called “synthetic pyrethroids,” having excellent insecticidal activity, and they are also useful as the intermediates of prostaglandin derivatives which serve as drugs.
For example, the compound of formula II below, an ester of 4hydroxy-3-methyl-2-(2-propynyl)cyclopent-2en-1-one with 2,2,3,3-tetramethylcyclopropanecarboxylic acid, is an excellent insecticide, having very strong knockdown activity and mortal activity (see, e.g., JP-B 50-15843/1975).
The cyclopentenolones of formula I include two kinds of optical isomers because they have an asymmetric carbon atom at position 4. In the case of synthetic pyrethroids containing such optical isomers as the alcohol components, it is well known that the difference in optical isomerism between these alcohol components makes a great difference in their insecticidal effects. For example, the compound of formula II above has been found to exhibit several times as excellent insecticidal activity in the case of an ester of (S)-4-hydroxy-3-methyl-2-(2-propynyl)cyclopent-2-en-1-one as in the case of an ester of the corresponding (R)-4-hydroxy-3-methyl-2-(2-propynyl)cyclopent-2-en-1-one.
Furthermore, similar optically active cyclopentenolones of formula I, such as 4-hydroxy-2-(7-hydroxyheptyl)-2-cyclopentenone, 4hydroxy-2-(6-methoxycarbonylhexyl)-2-cyclopentenone and 4hydroxy-2-(2-propenyl)-2-cyclopentenone, are useful as the intermediates of prostaglandin derivatives which serve as drugs.
For these reasons, there has been a great demand for the development of a method for separating and obtaining the optical isomers of cyclopentenolones of formula I as the intermediates of drugs, agricultural chemicals or other active products in an industrially favorable manner. In addition, for this purpose, in order to prepare a microorganism, for example, by a gene engineering technique, which microorganism can produce an excellent esterase capable of acting upon an organic carboxylic acid ester of a cyclopentenolone of formula I for asymmetric hydrolysis of the ester, the search of a gene coding for such an esterase has also been eagerly desired.
SUMMARY OF THE INVENTION
Under these circumstances, the present inventors have extensively studied and found an esterase gene coding for an esterase capable of acting upon an organic carboxylic acid ester of a cyclopentenolone of formula I for asymmetric hydrolysis of the ester to produce the cyclopentenolone in (R)-form with high optical purity, thereby completing the present invention.
Thus, the present invention provides:
1) An isolated esterase gene coding for an esterase capable of causing asymmetric hydrolysis of an organic carboxylic acid ester of a cyclopentenolone of formula I:
wherein R
1
is hydrogen or methyl, and R
2
is C
1
-C
10
alkyl C
2
-C
10
alkenyl, C
2
-C
10
alkynyl, C
1
-C
4
haloalkyl, a C
5
-C
9
hydroxy aliphatic hydrocarbon moiety which may be optionally protected on the terminal hydroxyl group thereof, or a C
5
-C
9
fatty acid residue which may be optionally protected on the terminal carboxyl group thereof, to produce the cyclopentenolone of formula I in (R)-form, and hybridizing to the base sequence of SEQ ID NO:1 (hereinafter referred to as the present gene);
2) the isolated esterase gene according to item 1, wherein the homology of the gene to the base sequence of SEQ ID NO:1 is 90% or higher.
3) The isolated esterase gene according to item 1, having a base sequence coding for the amino acid sequence of SEQ ID NO:2.
4) The isolated esterase gene according to item 1, having the base sequence of SEQ ID NO:1.
5) A plasmid containing the isolated esterase gene of item 1, 2, 3 or 4 (hereinafter referred to as the present plasmid).
6) A transformant obtained by transformation with the plasmid of item 5 (hereinafter referred to as the present transformant).
7) The transformant according to item 6, which is a microorganism.
8) An esterase produced by a microorganism having the isolated esterase gene of item 1, 2, 3 or 4 (hereinafter referred to as the present esterase).
9) The esterase according to item 8, wherein the microorganism having the isolated esterase gene of item 1, 2, 3 or 4 is the transformant of item 6.
10) A process for producing en esterase, which comprises cultivating the transformant of item 6 to produce an esterase capable of causing asymmetric hydrolysis of an organic carboxylic acid ester of a cyclopentenolone of formula I:
wherein R
1
is hydrogen or methyl, and R
2
is C
1
-C
10
alkyl, C
2
-C
10
alkenyl, C
2
-C
10
alkynyl, C
1
-C
4
haloalkyl, a C
5
-C
9
hydroxy aliphatic hydrocarbon moiety which may be optionally protected on the terminal hydroxyl group thereof, or a C
5
-C
9
fatty acid residue which may be optionally protected on the terminal carboxyl group thereof, to produce the cyclopentenolone of formula I in (R)-form hereinafter referred to as the present production process).
11) A method for the optical resolution of a cyclopentenolone of formula I:
wherein R
1
is hydrogen or methyl, and R
2
is C
1
-C
10
alkyl, C
2
-C
10
alkenyl, C
2
-C
10
alkynyl, C
1
-C
4
haloalkyl, a C
5
-C
9
aliphatic hydrocarbon moiety which may be optionally protected on the terminal hydroxyl group thereof, or a C
5
-C
9
fatty acid residue which may be optionally protected on the terminal carboxyl group thereof, which comprises allowing the esterase of item 8 to act upon an organic carboxylic acid ester of the cyclopentenolone of formula I for asymmetric hydrolysis of the ester; and separating the cyclopentenolone of formula I in (R)-form from the ester of the corresponding enantiomer thereof.
12) The optical resolution method according to item 11, wherein the cyclopentenolone of formula I is 4hydroxy-3-methyl-2-(2-propenyl)cyclopent-2-en-1-one.
13) The optical resolution method according to item 11, wherein the cyclopentenolone of formula I is 4hydroxy-3-methyl-2-(2-propynyl)cyclopent-2-en-1-one.


REFERENCES:
patent: 4607013 (1986-08-01), Mitsuda et al.
patent: 5290694 (1994-03-01), Nakanishi et al.
patent: 5306636 (1994-04-01), Iizumi et al.
patent: 0487541 (1992-06-01), None
patent: 0657535 (1995-06-01), None
patent: 6153965 (1994-06-01), None
patent: 07504561 (1995-05-01), None
Jorgensen et al., Journal of Bacteriology, vol. 173, No. 2, 559-567 (1991).
Sugihara et al., J. Biochem. 112, 598-603 (1992).
Kinoshita et al. Factors influencing enantioselectivity of lipase-catalyzed hydrolysis. Tetrahedron 52 (15): 5397-5406 (1996).
Hirose et al. Inversion of enantioselectivity in hydrolysis of 1, 4-dihydropyridines by point mutation of lipase PS. Tetrahedron Lett. 36(7): 1063-1066 (1995).
Sih et al. Differences in reactivity and enantioselectivity in Lipase reactions with carboxylic esters and alcohols bearing the same stereogenic center. Tetrahedron:Assymetry 6: 357-360 (1995).
Schrag et al.,Structure, vol. 5, No. 2, pp. 187-202 (1997).
Frenken et al.,Applied and Environmental Microbiology, vol. 58, No. 12, pp. 3787-3791 (Dec. 1992).

Affiliated with

Also associated with

Rating

Esterase gene and its use does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Esterase gene and its use, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Esterase gene and its use will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2993158