Effects of Detergents on Activity, Thermostability and Aggregation of Two Alkalithermophilic Lipases from Thermosyntropha lipolytica

Abstract

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 v(max) of both LipA and LipB were 12.6 U.mg(-1) and 13.3 U.mg(-1) and K(0.5) were 1.8 mM and 1.65 mM, respectively at 96 degrees C and pH(opt) (25 masculineC)of 9.4-9.6. In the presence of 0.2% SDS, the v(max) increased to 105 U.mg(-1) and 112 U.mg(-1), and K(0.5) values decreased to 200 microM and 140 microM 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: Alkalithermophilic; Lipases; SDS; non ionic- anionic and cationic detergents.; Thermosyntropha lipolytica; aggregation/oligomerization.

Fig. (1)

Effects of various detergents on the activity of LipA (○) and LipB (□). Increasing concentrations of the following detergents were used; SDS (A), Na cholate (B), CTAB (C), Triton X-100 (D), Tween 20 (E) and Tween 80 (F). Assays were carried out as described in the materials and methods section with some modifications. The assay buffer was 100 mM TAPS adjusted at pH^25°C9.0. The assay temperature was 92°C. Non ionic detergents at concentrations higher than their CMC values caused turbidity in the assay solution, thus, assays were centrifuged and diluted before measuring the absorption.

Fig. (2)

The hydrolysis rate of p-nitrophenyl laurate by LipA in the absence (●) and presence of 0.2% SDS (○). Assays were performed as described in the materials and methods section. Each assay contained 10 µg of freshly purified LipA. The assay duration was 15 min, at the end of the assay; reactions were stopped in ice. Since LipA and LipB look very much the same, LipB was omitted for clarity.

Fig. (3)

Temperature activity profiles in the presence of 0.2% SDS and absence of SDS (Insert). Assays, as described in the materials and methods section, were performed in triplicates at the pH optima of LipA (○) and LipB (□). All data were recorded as the difference between the assays and their controls.

Fig. (4)

The elution profiles of aggregated LipA (a) and LipB (b) after gel filtration chromatography. The first peaks (dotted line) represent aggregated enzymes eluted in the absence of SDS, the second sharper peak (solid line) represent monomeric enzyme treated and eluted with 1% SDS. The elution volume and corresponding molecular mass as calculated from standard marker is indicated on top of each peak. Elution volumes of molecular weight markers used for calculating the apparent molecular weight of the lipases were: Ferritin (440 kDa); 27.9 ml; bovine serum albumin (66 kDa), 35.56 ml; albumin (45 kDa), 36.8 ml; carbonic anhydrase (29kDa), 39.94 ml; and cytochrome (12.8 kDa), 41.67 ml.

Fig. (5)

Thermostability profiles of LipA (A) and LipB (B) in the absence and presence of detergents A15µg aliquots of LipA and LipB were incubated at 75°C in the presence of 1% (v/v) Tween 20 (■) and 1% (w/v) SDS (♦),and without detergent addition (control) (○). Lipase assays were conducted as described in the methods section.