Novel S-enantioselective lipase TALipB from Trichosporon asahii MSR54: Heterologous expression, characterization, conformational stability and homology modeling
•TALipB was cloned into pET22b (C-His tag), pET28a (N and C-His tags) and pEZZ18 (N-ZZ tag) vectors.•ZZ-tagged enzyme showed comparatively higher pH and temperature stability.•All the enzymes were mid to long chain specific and S-enantioselective, no effect of tags was observed in case of substrate specificity.•Tags effects the thermostability and catalytic efficiency of the enzymes, as ZZ-tagged enzyme was comparatively more thermostable and less catalytically efficient than other two.•3D model built by PHYRE showed high structural homology with Candida antarctica lipase B.
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 (254 mM−1 min−1) on p-NP caprate followed by HLipBH and ZZLipB. However, ZZLipB demonstrated best pH stability (pH 6–10), thermostability (t1/2 of 50 min at 70 °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 β-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.
Journal: Enzyme and Microbial Technology - Volume 83, February 2016, Pages 29–39