Monoacylglycerol lipase from moderately thermophilic Bacillus sp strain H-257: Molecular cloning, sequencing, and expression in Escherichia coli of the gene

Monoacylglycerol lipase [MGLP, EC 3.1.1.23] is produced intracellularly by the moderately thermophilic Bacillus sp. strain H-257, The gene encoding MG LP was cloned, sequenced, and expressed in Escherichia coli, A genomic libr ary of Bacillus sp. strain H-257, prepared in the plasmid vector pACYC184, was screened with a 0.2-kbp DNA fragment amplified by the polymerase chain reaction (PCR) with oligonucleotide primers designed based on the amino aci d sequence of a purified MGLP, The plasmid pMGLP31, identified by hybridiza tion with the amplified DNA fragment, contained a 5.3-kbp insert from Bacil lus sp. strain H-257 DNA, Sequence analysis of the MGLP gene revealed an op en reading frame encoding MGLP consisting of 250 amino acids, with a calcul ated molecular mass of 27.4 kDa, The deduced amino acid sequence of MGLP co ntained the consensus pentapeptide (-Gly-Xaa-Ser-Xaa-Gly-), which is conser ved among lipases, esterases, and serine proteases, The MGLP is homologous to a putative esterase/lipase from Streptomyces coelicolor (41.8% homology) , When pMGLP31 was introduced into E. coli DH1, the transformants produced MGLP intracellularly as an active form to an approximately 13.8-fold greate r extent than Bacillus sp. strain H-257, The purified recombinant MGLP was shown to be identical to the native enzyme in terms of chromatographic beha vior, isoelectric point, and physicochemical and catalytic properties.