Effects of urea and sodium dodecylsulphate on the stability profile of Thermomyces lanuginosus lipase

Abstract

The thermostability property of Thermomyces lanuginosus lipase (TLL) has been demonstrated but its resistance to protein denaturants has not been fully characterized. Therefore, this study investigated the effects of protein denaturants, urea, and sodium dodecyl sulphate (SDS), on TLL activity. Reaction mixtures containing 0.1 M Glycine-NaOH buffer, 0.001 µM TLL, and appropriate concentrations of protein denaturants were pre-incubated at 37 oC for 10 minutes. Reactions were initiated by the addition of appropriate concentration of substrate, para-nitrophenyl dodecanoate (pNPD), for a specific time interval and were terminated by the addition of 0.5 M TCA and 0.5 M NaOH. TLL activity was determined spectrophotometrically at 405 nm by monitoring the rate of hydrolysis of pNPD against a blank of the buffered substrate. TLL was active over a pH range of 3.0 to 9.0 and its activity was optimal at pH 9.0. However, TLL activity dropped by 41% at pH 12.0. TLL activity increased progressively with increase in temperature from 20 °C to 60 °C. Between 70 and 100 °C, a slight decrease in TLL activity was observed as compared to the activity at 60 °C. TLL activity was stable at pre-incubation temperatures ranging from 30 °C to 80 °C. Urea at 0.1 – 4.0 mM increased TLL activity. SDS at 0.5 – 4.0 mM increased TLL activity in a concentration-dependent manner with optimal activity obtained at 4.0 mM. Urea at 0.1, 1.0, 2.0 and 4.0 mM decreased maximum reaction rate (Vmax), catalytic constant (Kcat) and Michaelis constant (Km) of TLL, while SDS at 0.1 mM decreased Vmax, Kcat and Km. 1.0 mM SDS did not affect Vmax and Kcat of TLL, but reduced Km by 50%. At 2.0 mM SDS, Vmax, and Kcat increased by 43% while Km reduced. However, 4.0 mM SDS reduced Vmax, Kcat, and Km of TLL. In conclusion, findings from this study suggest that TLL partially and strongly resists denaturation by optimal concentrations of urea and SDS, respectively.