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 MGLP was cloned, sequenced, and expressed in Escherichia coli. A genomic library 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 acid sequence of a purified MGLP. The plasmid pMGLP31, identified by hybridization with the amplified DNA fragment, contained a 5.3-kbp insert from Bacillus sp. strain H-257 DNA. Sequence analysis of the MGLP gene revealed an open reading frame encoding MGLP consisting of 250 amino acids, with a calculated molecular mass of 27.4 kDa. The deduced amino acid sequence of MGLP contained the consensus pentapeptide (-Gly-Xaa-Ser-Xaa-Gly-), which is conserved 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 greater extent than Bacillus sp. strain H-257. The purified recombinant MGLP was shown to be identical to the native enzyme in terms of chromatographic behavior, isoelectric point, and physicochemical and catalytic properties.
Kitaura
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S
Diagnostics Research and Development Department, Diagnostics Division, Asahi Chemical Industry Co., Ltd., Mifuku, Ohito, Shizuoka 410-2321, Japan. kitaura.sb@om.asahi-kasei.co.jp
Suzuki
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K
Imamura
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S
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008994:Monoacylglycerol Lipases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; D008994:Monoacylglycerol Lipases