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

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...

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Veröffentlicht in:Enzyme and microbial technology 2001-10, Vol.29 (6), p.363-371
Hauptverfasser: Lee, Dong-Woo, Kim, Hack-Woo, Lee, Keun-Wook, Kim, Byoung-Chan, Choe, Eun-Ah, Seung Lee, Han, Kim, Doo-Sik, Pyun, Yu-Ryang
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Sprache:eng
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Zusammenfassung: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 Cu 2+, Hg 2+, and Co 2+. In contrast, BTID-B was slightly activated by Ca 2+, Co 2+, and Mn 2+ 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 K m values of BTID-A and BTID-B for the hydrolysis of tricaprylin were 1.82 mM ( V max , 12.8 μmol min −1 mg −1) and 6.24 mM ( V max , 63.3 μmol min −1 mg −1), respectively.
ISSN:0141-0229
1879-0909
DOI:10.1016/S0141-0229(01)00408-2