Malathion carboxylesterase

Details Malathion carboxylesterase Malathion carboxylesterase

1998-05-20

2001-05-22

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S183000, C435S069100, C435S252300, C435S320100, C536S023200

Reexamination Certificate

active

06235515

ABSTRACT:

This invention relates to an enzyme (and the nucleic acid sequences encoding this enzyme), termed malathion carboxylesterase (MCE) which is able to efficiently hydrolyse a specific class of organophosphate (OP) insecticides which have the general structures:
(eg. malathion, phenthoate)
(eg. malaoxon, phenthoate oxon)
where X contains one or more carboxylester groups for thion type organophosphates but is unconstrained for oxon type organophosphates.
Residues of organophosphate insecticides are undesirable contaminants of the environment and a range of commodities. Areas of particular sensitivity include contamination of domestic water supplies and soil, residues above permissible levels in various food and fibre exports and contamination of domestic pets. Bioremediation strategies are therefore required for eliminating or reducing these insecticide residues. One proposed strategy involves the use of enzymes capable of immobilising or degrading the insecticide residues. Such enzymes may be employed, for example, in bioreactors through which contaminated water could be passed; in production animal dips to reduce problems with contaminated pasture and run off into water supplies; or in washing solutions after post harvest disinfestation of fruit, vegetables or animal products to reduce residue levels and withholding times. Suitable enzymes for degrading pesticide residues include esterases. It is desirable that the esterases be relatively specific and hydrolyse the pesticide residues at a rapid rate.
The MCE enzyme has been purified from different malathion resistant strains of
L. cuprina
, RM and der-L (Whyard S., Russell R. J. and Walker V. K., Biochemical Genetics 32: 9, 1994; Whyard S. and Walker V. K., Pesticide Biochemistry and Physiology 50: 198, 1994). It is a 60.5 kDa monomer with a K
m
for malathion of 11.0±0.4 &mgr;M and a V
max
of 775±28 nmol malathion/min/mg. It also has a high turnover rate for malathion (k
cat
=46 min
−1
).
In order to enable the production of useful amounts of the MCE enzyme the present inventors sought to clone the putative MCE gene from a malathion resistant strain of
L. cuprina
(RM-
8
) using PCR and cloning techniques.
The MCE gene in
L. cuprina
has been mapped using classical genetic techniques to a position within 0.7 map units from the E
3
gene on chromosome
4
. The likely homologue of MCE in
Drosophila melanogaster
, Mce, has been mapped to the right arm of chromosome
3
in the vicinity of the genes encoding the major &agr;-carboxylesterase, EST 9, and the orthologue of
L. cuprina
E
3
, EST23 (Spackman M. E., Oakeshott J. G., Smyth K-A., Medveczky K. M., and Russell R. J., Biochemical Genetics, 32: 39, 1994).
In order to clone the MCE gene from
L. cuprina
, it was decided to use the wealth of molecular genetic techniques available for
D. melanogaster
to clone the MCE homologue and use these clones as probes to isolate the
L. cuprina
genes themselves.
In summary, five esterase amplicons were isolated from
L. cuprina
genomic and cDNA. Four of the five
L. cuprina
amplicons obtained by PCR using cluster specific primers were designated Lc&agr;E
7
, Lc&agr;E
8
, Lc&agr;E
9
and Lc&agr;E
10
on the basis of homology to the corresponding Drosophila genes. The fifth, Lc#
53
, could not be assigned with any confidence on the basis of similarity to any of the Drosophila genes.
MCE specific activity is highest in the adult head, rather than the thorax or abdomen (Smyth,K-M., Walker,V. K., Russell,R. J. and Oakeshott,J. G. Pesticide Biochemistry and Physiology, 54:48, 1996). On this basis, Lc&agr;E
7
, Lc&agr;E
8
and Lc&agr;E
10
were all MCE candidates. Previous physiological studies of Parker,A. P., Russell,R. J., Delves,A. C. and Oakeshott,J. G.(Pesticide Biochemistry and Physiology 41:305, 1991) have shown that the E
3
(Lc&agr;E
7
) enzyme is present in the adult head. Moreover, the Lc&agr;E
8
and Lc&agr;E
0
genes are also expressed in the head since PCR using cluster-specific primers were able to amplify these genes from a head cDNA library. PCR failed to detect Lc&agr;E
9
and Lc#
53
in either larval fat body or adult head cDNA and Northern analysis of the
D. melanogaster
&agr;E9 homologue indicated that this gene was only expressed in embryos. Therefore both Lc&agr;E
9
and Lc#
53
were discounted as candidates for the genes encoding E
3
and MCE.
The Lc&agr;E
8
and Lc&agr;E
10
genes were initially chosen as prime MCE candidates on the basis of this distribution and due to the fact that it was known that Lc&agr;E
7
encodes the E
3
enzyme involved in diazinon/parathion OP resistance in
L. cuprina
(PCI/AU 95/00016: “Enzyme based bioremediation”) and it was thought that malathion resistance and diazinon/parathion resistance were encode by separate genes.
The present inventors have made the surprising finding that it is a variant of Lc&agr;E
7
which encodes the MCE enzyme. This gene has been expressed in vitro and the product shown to have MCE activity. The expressed product can be formulated for use in degrading environmental carboxylester or dimethyl general OPs.
Accordingly, in a first aspect, the present invention consists in an isolated DNA molecule encoding an enzyme capable of hydrolysing at least one organophosphate selected from the group consisting of carboxylester organophosphates and dimethyl-oxon organophosphates, the DNA molecule comprising a nucleotide sequence having at least 60%, preferably at least 80% and more preferably at least 95% homology with Lc&agr;E
7
, in which the protein encoded by the DNA molecule differs from E
3
at least in the substitution of Trp at position
251
with an amino acid selected from the group consisting of Leu, Ser, Ala, Ile, Val, Thr, Cys, Met and Gly.
In a preferred embodiment the present invention the isolated DNA molecule has a sequence as shown in FIG. 1 or a sequence which hybridises thereto with the proviso that the protein encoded by the DNA molecule differs from E
3
at least in the substitution of Trp at position
251
with an amino acid selected from the group consisting of Leu, Ser, Ala, Ile, Val, Thr, Cys, Met and Gly.
In a preferred embodiment of the present invention the Trp at position
251
is substituted with Ieu or Ser.
As is stated above the present invention includes nucleic acid molecules which hybridise to the sequence shown in FIG.
1
. Preferably such hybridisation occurs at, or between, low and high stringency conditions. In general terms, low stringency conditions can be defined as 3xSCC at about ambient temperature to 65° C., and high stringency conditions as 0.1xSSC at about 65° C. SSC is the abbreviation of a buffer of 0.15M NaCl, 0.015M trisodium citrate. 3xSSC is three times as strong as SSC and so on.
In a second aspect the present invention consists in an isolated DNA molecule, the DNA molecule encoding a polypeptide having the amino acid sequence of RM-
8
Con shown in
FIG. 1
or the amino acid sequence of Md&agr;E
7
shown in
FIG. 3
in which Trp at position
251
is replaced with Ser.
Homologues of the MCE encoding sequence may also be present in the genome of other insects, and particularly other species of Diptera. Thus, it is to be understood that the invention also extends to these homologues An example of this is provided by the results set out hereunder regarding Musca MCE.
The isolated DNA molecules of the present invention may be cloned into a suitable expression vector and subsequently transfected into a prokaryotic or eukaryotic host cell for expression of the enzyme. A particularly suitable system involves baculovirus vectors and an insect cell line.
In a third aspect the present invention consists in a method of producing an enzyme capable of hydrolysing at least one organophosphate selected from the group consisting of carboxylester organophosphates and dimethyl-oxon organophosphates, or an enzymatically active portion thereof, the method comprising transforming a host cell with the DNA molecule of the first aspect of the present invention operatively linked to a control seq

Affiliated with

Also associated with

Rating

Malathion carboxylesterase does not yet have a rating. At this time, there are no reviews or comments for this patent.

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

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2450255