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 |
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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. |
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ISSN: | 0141-0229 1879-0909 |
DOI: | 10.1016/S0141-0229(01)00408-2 |