2000 年 127 巻 3 号 p. 419-425
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-4 B, 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°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°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 Km values for the hydrolysis of 1-monolauroylglycerol, 1-monooleoylglycerol, and 2-monooleoylglycerol were determined to be 140, 83 and 59 μM, 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.
