TALipB (33 kDa) is a solvent stable, enantioselective lipase from Trichosporon asahii MSR54. It is cysteine-rich and shows activation in presence of thiol reducing agents. DIANNA server predicted three disulphide bridges C53-C195 (S1), C89-C228 (S2) and C164-C254 (S3) in the enzyme. Selective disruption of disulphide bonds by cysteine to alanine mutations at Cys53 and Cys89 of S1 and S2 bonds resulted in enzyme activation. Mutant mTALipB (S1+S2) showed increase in specific activity by similar to 4-fold (834 mM/mg) and improved V-max of 6.27 mu mol/ml/min at 40 C degrees on pNP caprate. Temperature optima of mTALipB shifted from 50 to 40 degrees C and activation energy decreased by 0.7 kcal mol(-1). However, the mutant was less thermostable with a t(1/2) of 18 min at 60 degrees C as compared to t(1/2) of 38 min for the native enzyme. Mutant also displayed an improved activity on all pNP esters and higher enantiomeric excess (61%) during esterification of (+/-) 1-phenylethanol. Far-UV CD analysis showed significant changes in secondary structure after S-S bridge disruption with 7.16% decrease in alpha-helices and 1.31% increase in beta-sheets. In silico analysis predicted two lids (alpha 5 and alpha 9) in TALipB. Molecular dynamic simulations at 40 degrees C and 50 degrees C revealed that in the mTALipB, both the lids opened at 40 degrees C with clockwise and anticlockwise rotations in Lid1 and Lid2, respectively. In the native protein, however, the lid was only partially open even at 50 degrees C. Concomitant to lid flexibility, there was an extension of accessible catalytic triad surface area resulting in improved catalytic efficiency of the mutant enzyme. (C) 2016 Elsevier Inc. All rights reserved.