Substrate kinetics of thiol activated hyperthermostable alkaline lipase of Bacillus sonorensis 4R and its application in bio-detergent formulation

The substrate kinetics of a hyperthermostable alkaline lipase from Bacillus sonorensis 4R (BSL) was studied and the enzyme was used to develop presoak formulation for removing oil stains at 37 °C and 80 °C. The purified BSL was thiol activated with 112.49% and 37.49% enhancement in presence of 5 mM thiourea and dithiothreitol (DTT) respectively. BSL displayed higher specificity for mustard oil and cotton seed oil cake than olive oil as a standard substrate. The maximal velocity (Vmax) and Michaelis constant (Km) values determined in presence of olive oil, mustard oil and cotton seed oil cake were 5.0, 7.14, 5.55 µM and 3.22, 2.5 and 2.22 µM/ml/min respectively. Effect of phenylmethylsulfonyl flouride (PMSF) and N-Bromosuccinimide (NBS) on BSL catalyzed hydrolysis of olive oil, mustard oil and cotton seed oil cake were studied in terms of changes in Vmax, Km, catalytic efficiency and substrate utilization capacity. BSL catalyzed improved hydrolysis of cotton seed oil cake in presence of thiourea and retained 100% stability in presence of non-polar solvents, surfactants and detergents. BSL (15 Eu/100 ml) was effective in removal of olive oil stains studied at three aging periods (0, 7 and 15 d) and two washing temperatures (37 °C and 80 °C). The BSL action during wash performance studies for all aging periods was demonstrated clearly by quantitated fatty acid release. BSL exhibited excellent olive oil removal capacity than buffer (pH 9.0) and Tide (1%). The combination of BSL and Tide proved best formulation for stain removal of olive oil soiled fabrics.