title: Purification and characterization of a monoacylglycerol lipase from the moderately thermophilic Bacillus sp. H-257.
abstract: A thermostable monoacylglycerol lipase [MGLP, EC 3.1.1.23] was purified for the first time from a cell-free extract of the moderately thermophilic Bacillus sp. H-257. The enzyme was purified 3,028-fold to homogeneity by chromatography using Octyl-Sepharose CL-4B, Q-Sepharose FF, and Superose 12 columns. The molecular mass of the MGLP was estimated to be 25 kDa by gel filtration and 24 kDa by SDS-PAGE, suggesting a monomeric protein. The isoelectric point was determined to be 4.66 by isoelectric focusing. The MGLP retained its full activity upon incubation at 60 degrees C for 10 min (pH 7. 3), and was stable at pH 7-10. The optimal temperature for activity at pH 7.5 was 75 degrees C, and the maximum activity was observed from pH 6-8. This enzyme hydrolyzes monoacylglycerols, with the highest activity occurring with 1-monolauroylglycerol. Di- and triacylglycerols, on the other hand, are essentially inert as substrates for the enzyme. The K(m) values for the hydrolysis of 1-monolauroylglycerol, 1-monooleoylglycerol, and 2-monooleoylglycerol were determined to be 140, 83 and 59 mM, respectively. The enzyme was not inhibited by cholate, but was slightly inhibited by Triton X-100 and deoxycholate. The amino acid sequence of the N-terminal region of the enzyme (16 residues) was also determined.
authors.affiliation: Diagnostic Research and Development, Diagnostic Division, Asahi Chemical Industry Co., Ltd., Mifuku, Ohito, Shizuoka 410-2321, Japan. QZP06502@mifty.ne.jp
authors.forename: S
authors.initials: S
authors.lastname: Imamura
authors.forename: S
authors.initials: S
authors.lastname: Kitaura
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D002384:Catalysis; D002847:Chromatography, Agarose; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008994:Monoacylglycerol Lipases / Q000737:chemistry* / Q000302:isolation & purification*; D013696:Temperature
chemical_list: D008994:Monoacylglycerol Lipases