Characterization of a Novel Thiol Activated Phospholipase TAPLB1 from Trichosporon Asahii MSR 54

Phospholipases are hydrolytic enzymes that play crucial roles in vivo and also possess immense biotechnological potential. In the present study, the phospholipase B of Trichosporon asahii MSR54 was overexpressed in E. coli and characterized. The 68-kDa enzyme was monomeric in solution and possessed phospholipase, lysophospholipase, esterase and acyltransferase activities. It was maximally active at pH 8.0 and 40 °C. The enzyme retained >50% activity between pH 3.0-8.0 and had a half-life of 30 min at 60 °C. Its activity was not metal dependent and was stable in the presence of most metal ions. Its catalytic efficiency on lysophosphatidyl choline was 1.0 × 10 mM h. Site directed mutagenesis revealed R (present in the GYRAMV motif), S (present in the conserved GLSGG motif) and D (present in LVDXGE motif) to be the crucial amino acid residues for esterolytic activity. S and D were also the catalytic amino acids for lysophospholipase and phospholipase activities of the enzymes, while R was not involved in catalysis of phospholipid substrates. Further, it was found that cysteine residues in C and C were involved in disulphide linkages that imparted the properties of thiol activation and thermostability, respectively.