CDNA encoding plant-derived epoxide hydrolase, gene encoding...

Details CDNA encoding plant-derived epoxide hydrolase, gene encoding... CDNA encoding plant-derived epoxide hydrolase, gene encoding...

1999-10-06

2002-04-16

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S252300, C435S320100, C530S350000, C536S023200

Reexamination Certificate

active

06372469

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a cDNA encoding a plant-derived hydrolase, a gene encoding the same, a plasmid vector carrying said cDNA and a transformant.
BACKGROUND OF THE INVENTION
Epoxide hydrolase is an enzyme involved in the biological protective system functioning for preventing chemical reactions hazardous for living organisms in such a manner that the enzyme hydrolyses hazardous epoxide compounds generated through peroxidation in living organisms, to eliminate the high chemical reactivity of the epoxide compounds.
It is evidenced that epoxide hydrolase is present in animal cells and plant cells. Particularly, epoxide hydrolase derived from plants is expected to be applicable to agricultural fields and food industries. Because the content thereof is very low, the mass-scale production thereof has been believed to be extremely difficult.
As to genetic information concerning epoxide hydrolase, currently, reports have been issued about the genetic information thereof from animal cells, such as human liver cell [Beetham, J. K. et al., Arch. Biochem. Biophys., 305, 197-201 (1993)] and liver cells from rats and mice [Knehr, M. et al., J. Biol. Chem., 268, 17623-17627 (1993); Grant, D. F. et al., J. Biol. Chem., 268, 17628-17633 (1993)].
Meanwhile, the genetic information thereof from plant cells including potato [Stapleton, A. et al., Plant J. 6, 251-258 (1994)] and Arabidopsis [Kiyosue, T. et al., Plant J., 6, 259-269 (1994)] has been reported as well.
However, no report regarding genetic information about epoxide hydrolase derived from plants has been published yet, except the aforementioned reports. No plant-derived epoxide hydrolase with high activity has been reported yet.
SUMMARY OF THE INVENTION
The inventors have made attempts to purify a plant-derived epoxide hydrolase and to clone the cDNA and gene of the epoxide hydrolase based on the amino acid sequence thereof. Consequently, the inventors have successfully identified the whole structure of the cDNA of the enzyme and that of the gene thereof and have also achieved successfully the construction of an expression system in
Escherichia coli
by utilizing the cDNA and the gene. Thus, the invention has been achieved.
Based on the analysis of the amino acid sequence of the purified epoxide hydrolase, synthetic degenerate oligonucleotides were prepared. By subsequently extracting MRNA from a fully ripened soy seed and synthesizing a double-stranded cDNA using the mRNA, which was then integrated via an adapter into a phage vector, a cDNA library was constructed.
By labeling the prepared synthetic oligonucleotides and using the labeled synthetic oligonucleotides as probes, cDNA encoding the epoxide hydrolase of its full length was cloned by screening from the aforementioned cDNA library. The analysis of the primary structure of the cDNA indicated the whole amino acid sequence of the epoxide hydrolase including the signal peptide.
The epoxide hydrolase gene was cloned by using a commercially available gene library prepared by partially digesting nuclear DNA extracted and purified from sprout-developed soy with a restriction endonuclease Mbo I and integrating the digestion product in a phage vector.
By labeling the preliminarily recovered cDNA of the epoxide hydrolase and using the labeled cDNA as probe for screening from the aforementioned gene library, the epoxide hydrolase gene including the 5′ upstream region was cloned.
Using the cloned epoxide hydrolase cDNA to construct an
Escherichia coli
expression system by utilizing a promoter T
7
, the present enzyme was successfully expressed while a simple and rapid purification method thereof was also established.
More specifically, a first aspect of the invention relates to a cDNA encoding the plant-derived epoxide hydrolase having the amino acid sequence of SQ ID No. 1 in the Sequence Listing.
A second aspect of the invention relates to a gene encoding the plant-derived epoxide hydrolase having the amino acid sequence of SQ ID No. 2 in the Sequence Listing.
A third aspect of the invention relates to a plasmid vector carrying the cDNA encoding the plant-derived epoxide hydrolase in the first aspect of the invention.
A fourth aspect of the invention relates to a transformant (FERM BP-6624) retaining the plasmid vector in the third aspect of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The invention will now be described in more detail hereinbelow.
As described in the column of “Description of the Related Art”, the epoxide hydrolase of the invention is an enzyme with an action hydrolyzing epoxide hazardous to living organisms to generate diol and the like.
The first aspect of the invention is now described.
The first aspect of the invention relates to the cDNA encoding the plant-derived epoxide hydrolase having the amino acid sequence of SEQ ID No. 1 in the Sequence Listing.
The inventors have succeeded to obtain the cDNA of the first aspect of the invention in the following manner.
1. Preparation of cDNA Library
1.1 Extraction and Purification of Epoxide Hydrolase in Plants
Epoxide hydrolase is present in cells of various plants and animals. In the first aspect of the invention, epoxide hydrolase derived from the cells of a plant is specifically used.
The cells of a plant are preferably cells of any seed with abundance of epoxide hydrolase, but are not limited thereto.
For extracting epoxide hydrolase from soy seed, use is preferably made of soy between early growing stage and ripe stage, preferably soy between early growing stage and later growing stage.
So as to obtain epoxide hydrolase, a raw material plant should necessarily be purified at a high level. The raw material plant includes for example soy, Arabidopsis, and potato.
The method for purifying the enzyme is illustrated in a case using soy as the raw material. Grinding the plant seed between early growing stage and ripe stage, and adding an appropriate buffer solution to the ground seed to extract the soluble fraction, the fraction is subjected to dialysis to separate the liquid fraction from the solid fraction. The resulting crude enzyme solution is applied to hydrophobic chromatography or gel filtration, whereby almost pure epoxide hydrolase can be recovered.
1.2 Analysis of Inner Amino Acid Sequence of Purified Epoxide Hydrolase
The purified epoxide hydrolase obtained in 1.1 cannot be analyzed as such of its inner amino acid sequence because the N terminus thereof is blocked. Therefore, the following procedures should be carried out.
Enzymatically degrading the epoxide hydrolase into short peptides and fractionating samples of the individual peptides by high performance liquid chromatography, the amino acid sequence of each of the samples is determined. The amino acid sequence is conveniently determined by Edman degradation using an automatic amino acid sequencer.
The individual samples are examined of their amino acid sequences; oligonucleotides are synthesized by using selected regions with less degeneracy among them; and the resulting oligonucleotides are used as the following probes.
1.3 Preparation of Poly(A)
+
RNA
RNA is then extracted from the plant seed, to prepare poly(A)
+
RNA from the extracted total RNA.
The poly (A)
+
RNA can be prepared from the plant by using the SDS-phenol method according to the method by Fukazawa, C. et al., Journal of Biological Chemistry, 200, 6234-6239 (1985).
1.4 Construction of cDNA Library
Synthetically preparing a double-stranded cDNA by using the resulting mRNA and integrating the cDNA via an adapter in a phage vector, a cDNA library is constructed.
The cDNA library can be prepared satisfactorily by various methods including for example the Okayama-Berg method [Okayama, H. and Berg, P., Mol. Cell Biol., 2, p. 161 (1982)] and the Gubler-Hoffman method [Gubler, U. and Hoffman, B. J., Gene, 25, p.263 (1983)]; and for simplicity, the latter method is preferable.
The Gubler-Hoffman method is now described below.
First, a double-stranded cDNA is synthetically

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