A novel lipase encoding gene, TALipB from Trichosporon asahii MSR54 was heterologously expressed in Escherichia coli using three vectors, pET22b, pET28a & pEZZ18. The three recombinant proteins, viz. C-hexahistidine fused HLipB, N and C-hexahistidine fused HLipBH and ZZ-fused ZZLipB were purified using affinity chromatography. All the three enzymes were mid to long fatty acyl chain selective on p-NP esters and S-enantioselective irrespective of tags. HLipB had lowest activation energy (3.5 Kcal mol(-1)) and highest catalytic efficiency (254mM(-1) min(-1)) on p-NP caprate followed by HLipBH and ZZLipB. However, ZZLipB demonstrated best pH stability (pH 6-10), thermostability (t(1/2) of 50 min at 70 degrees C) and stability toward the denaturant Guanidium chloride (300 mM). Far-UV CD and fluorescence studies confirmed the role of N-terminal ZZ-tag in stabilizing the protein by altering its secondary and tertiary structures. All the three proteins were thiol activated. ZZLipB required higher concentration of beta-mercaptoethanol as compared to the other two proteins to attain similar velocity. This indicated the involvement of additional disulfide bonds in its conformational stability. In silico analysis suggested low sequence identity of the enzyme with the available database but a close structural homology with Candida antarctica lipase B (CALB) was revealed by PHYRE2. MULTALIN with CALB predicted the active site residues (Ser137-Asp228-His261) which were confirmed by superimposition and site directed mutagenesis. (C) 2015 Elsevier Inc. All rights reserved.