Department of Microbiology, University of Georgia, Athens, Georgia 30602-2605, USA
Thermosyntropha lipolytica DSM 11003, an anaerobic thermophilic lipolytic bacterium, produces the two alkalithermophilic lipases, LipA and LipB. Among all tested detergents, the two lipases were mostly affected by SDS when used at concentrations below its critical micelle concentration (CMC). In the absence of SDS, the vmax of both LipA and LipB were 12.6 U·mg-1 and 13.3 U·mg-1 and K0.5 were 1.8 mM and 1.65 mM, respectively at 96°C and pHopt25ºCof 9.4-9.6. In the presence of 0.2% SDS, the vmax increased to 105 U·mg-1 and 112 U·mg-1, and K0.5 values decreased to 200 µM and 140 µM for LipA and LipB, respectively. Inhibitory assays of lipases using diisopropyl p-nitrophenylphosphate (E600) with increasing concentration of SDS and Tween 20 strongly suggest that SDS and Tween 20 do bind to the lid domain and/or active site pocket, thus promoting conformational changes that facilitate active site accessibility for the substrate. The two lipases exhibited moderate activation in the presence of nonionic detergents when used below their CMC values. Both lipases were found to exhibit strong tendency to aggregate as observed through gel filtration chromatography and gradient native gel electrophoresis. The addition of 1.0% (w/v) SDS led to disaggregation as the lipases were eluted corresponding to their monomeric mass (based on SDS gel electrophoresis value) and caused a significant decrease in thermostability, suggesting that, enzyme aggregation might be a major contributor to the high thermostability of LipA and LipB.
Keywords: Thermosyntropha lipolytica, Lipases, aggregation/oligomerization, Alkalithermophilic, SDS; non ionic- anionic and cationic detergents.
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