Isolation and heterologous expression of an epoxide hydrolase from Rhodotorula mucilaginosa.
Michel Labuschagne (1), Catherine Madzak (2), Jacobus Albertyn (1)
(1) Biotechnology, University of the Free State, Nelson Mandela Ave., Bloemfontein, 9300, South Africa; (2) UMR Microbiologie et Génétique Moléculaire INRA - CNRS - INAPG, CBAI, 78850 Thiverval Grignon, France.
Epoxide hydrolases (EHs) are the newest and most promising addition to the toolkit for the enantiomeric resolution of racemic epoxides. New potentially EH encoding genes need to be isolated and functionally expressed to complement this emerging technology. The EH encoding gene from R. mucilaginosa was isolated using degenerate primers in combination with inverse PCR. The genomic EH sequence revealed a 1979 bp sequence interrupted by 9 introns. RT-PCR revealed an ORF of 1185 bp encoding a predicted EH consisting of 394 amino acids. A yeast based system was used for functional heterologous expression of the EH encoding gene at levels exceeding the native activity. The recombinant EH exhibited some degree of substrate dependent enantioselectivity. Site directed mutagenesis based on sequence alignments revealed some useful information.