Purification and characterization of two distinct thermostable lipases, from the gram-positive thermophilic bacterium Bacillus thermoleovorans ID-1

The thermophilic bacterium Bacillus thermoleovorans ID-I can hydrolyze a va riety of oils such as olive oil, soybean oil, palm oil, and lard as a carbo n source (1.5%, v/v) after 72 h of culture at 50 degreesC. In this study, w e purified to homogeneity two distinct thermostable, lipases, designated BT ID-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 supernata nt 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, Resou rce PHE, and Mono Q column chromatography. Previously, the gene encoding BT ID-B of B. thermoleovorans ID-1 has been cloned, sequenced, and expressed i n Escherichia coli. Recombinant BTID-B was purified 108-fold from a cell ex tract of E. coli by heat precipitation, DEAE-Sepharose CL6B, and Sephacryl S200 column chromatography. Molecular mass of BTID-A was approximately 18 k Da and its activity was maximum at 60 to 65 degreesC. The pH optimum for BT ID-A was 9.0. On the other hand, BTID-B was a larger protein with a molecul ar 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, DM SO, and beta -mercaptoethanol, and divalent ions including Cu2+, Hg2+, and Co2+. In contrast, BTID-B was slightly activated by Ca2+, Co2+, and Mn2+ io ns and strongly resistant to organic solvents. Although both of the enzymes showed different substrate specificities, their maximal activities were fo und with tricaprylin (C8) as a substrate. The K-m values of BTID-A and BTID -B for the hydrolysis of tricaprylin were 1.82 mM (V-max, 12.8 mu mol min(- 1) mg(-1)) and 6.24 mM (V-max, 63.3 mu mol min(-1) mg(-1)), respectively. ( C) 2001 Elsevier Science Inc. All rights reserved.