A degradative bacterium, M6, was isolated and presumptively identified as Plesiomonas sp. strain M6 was able to hydrolyze methyl parathion to p-nitrophenol. A novel organophosphate hydrolase gene designated mpd was selected from its genomic library prepared by shotgun cloning. The nucleotide sequence of the mpd gene was determined. The gene could be effectively expressed in Esherichia coli.

The methyl parathion hydrolase (MPH)-encoding gene mpd was placed under the control of the P43 promoter and Bacillus subtilis nprB signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain B. subtilis WB800 as the host.

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identiWed. To further conWrm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was puriWed to homogeneity by metal-aYnity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the puriWed recombinant MPH indicated that a signal peptide of the Wrst 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the puriWed mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel Wltration of the puriWed mature recombinant MPH revealed that the MPH was a monomer.

α-L-Rhamnosidases play important roles in the metabolism of plant cell walls, glycosides and bacterial biofilms. This enzyme is also used industrially for debittering citrus fruits by releasing rhamnose from the plant flavonoid naringin. Bacillus sp. GL1 α-l-rhamnosidase (RhaB) is a member of glycoside hydrolase (GH) family 78. Native and selenomethionine-derivative enzymes were crystallized at 293 K by hanging-drop vapour diffusion with polyethylene glycol 8000 as a precipitant. This is the first report of the crystallization of a family GH78 enzyme.

A facilitative and efficient promoter-trapping vector, pUC-mpd, was constructed with the promoterless methyl parathion hydrolase gene as the reporter. This reporter gene is easily used to clone promoters with different promoting strength on selective plates. Promoter regions of the ytkA and ywoF genes with strong promoting and signal peptide functions were cloned from the Bacillus subtilis 168 genomic promoter library with this vector.