Selective disruption of disulphide bonds lowered activation energy and improved catalytic efficiency in TALipB from Trichosporon asahii MSR54: MD simulations revealed flexible lid and extended substrate binding area in the mutant.

Abstract

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 ∼4-fold (834 mM/mg) and improved Vmax of 6.27 μmol/ml/min at 40 °Con pNP caprate. Temperature optima of mTALipB shifted from 50 to 40 °C and activation energy decreased by 0.7 kcal mol(-1). However, the mutant was less thermostable with a t1/2 of 18 min at 60 °C as compared to t1/2 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 α-helices and 1.31% increase in β-sheets. In silico analysis predicted two lids (α5 and α9) in TALipB. Molecular dynamic simulations at 40 °C and 50 °C revealed that in the mTALipB, both the lids opened at 40 °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 °C. Concomitant to lid flexibility, there was an extension of accessible catalytic triad surface area resulting in improved catalytic efficiency of the mutant enzyme.