Journal of Biotechnology & Biomaterials
Open Access

Abstract

A novel lipase encoding gene, TALipB from Trichosporon asahii MSR54 was heterologously expressed in Escherichia coli using three vectors, pET22b, pET28a and pEZZ18. Purification was done using respective affinity chromatography as N-hexahistidine fused HLipB, N and C-hexahistidine fused HLipBH and ZZ-fused ZZLipB. Study showed that the enzyme was mid to long chain selective on p-NP esters and S-enantioselective irrespective of tags. Among these, HLipB had lowest activation energy (3.5 Kcal mol-1) and highest catalytic efficiency (254 mM-1min-1) on p-NP caproate followed by HLipBH and ZZLipB. However, ZZLipB demonstrated best pH stability (pH: 6-10), thermostability (t1/2: 70�º C for 50 min) and stability towards denaturants (GdmCl 500 mM and acrylamide 100 mM). Far-UV CD and fluorescence study showed that N-terminal ZZ-tag conferred stability by altering both secondary and tertiary structure. All the three proteins were thiol activated and structural changes during activation revealed that ZZLipB required higher concentration of BME to attain the similar velocity which indicated the involvement of additional disulfide bonds in its conformational stability. In silico analysis revealed that the enzyme had low identity with the available database. However Candida antarctica lipase B was identified as closest structural homolog using PHYRE2. MULTALIN with CALB predicted the active site residues (Ser137-Asp228-His261) which were confirmed by superimposition and site directed mutagenesis.

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