The thermophilic bacterium Bacillus thermoleovorans ID-1 can hydrolyze a variety of oils such as olive oil, soybean oil, palm oil, and lard as a carbon source (1.5%, v/v) after 72 h of culture at 50°C. In this study, we purified to homogeneity two distinct thermostable lipases, designated BTID-A (B. thermoleovorans ID-1 lipase A) and BTID-B (B. thermoleovorans ID-1 lipase B). BTID-A was purified 300-fold from a cell-free culture supernatant of B. thermoleovorans ID-1 grown in modified TYEM medium in the absence of a lipid substrate as an inducer. Purification of BTID-A was carried out by ammonium sulfate precipitation, DEAE-Sepharose CL6B, Superdex 200, Resource PHE, and Mono Q column chromatography. Previously, the gene encoding BTID-B of B. thermoleovorans ID-1 has been cloned, sequenced, and expressed in Escherichia coli. Recombinant BTID-B was purified 108-fold from a cell extract of E. coli by heat precipitation, DEAE-Sepharose CL6B, and Sephacryl S200 column chromatography. Molecular mass of BTID-A was approximately 18 kDa and its activity was maximum at 60 to 65°C. The pH optimum for BTID-A was 9.0. On the other hand, BTID-B was a larger protein with a molecular mass of 43 kDa, but showed the similar optima for its activity as BTID-A. The activity of BTID-A was inhibited by organic solvents such as EtOH, DMSO, and β-mercaptoethanol, and divalent ions including Cu2+, Hg2+, and Co2+. In contrast, BTID-B was slightly activated by Ca2+, Co2+, and Mn2+ ions and strongly resistant to organic solvents. Although both of the enzymes showed different substrate specificities, their maximal activities were found with tricaprylin (C8) as a substrate. The Km values of BTID-A and BTID-B for the hydrolysis of tricaprylin were 1.82 mM (Vmax, 12.8 μmol min-1 mg-1) and 6.24 mM (Vmax, 63.3 μmol min-1 mg-1), respectively.
Bibliographical noteFunding Information:
This work [985-1200-004-2] was supported by the Korea Science and Engineering Foundation (KOSEF). Technical support for this work by Prof. G. Antranikian and H. Märkl (Technical University of Hamburg-Harburg, Germany) is greatly appreciated. We express our gratitude to Dr. M.A. Yu for her help on preparing this paper.
All Science Journal Classification (ASJC) codes
- Applied Microbiology and Biotechnology