Presence of multiple acyltranferases with diverse substrate specificity in Bacillus smithii strain IITR6b2 and characterization of unique acyltransferase with nicotinamide

In the present study two acyltransferases, designated as acyltransferase A and acyltransferase B having completely different substrate specificities were observed in Bacillus smithii strain IITR6b2. Acyltransferase A was partially purified while acyltransferase B was purified to homogeneity by a four steps process with a purification fold of 12.1 and 37.5% yield. Acyltransferase A was found to have affinity for only short chain aliphatic amides with maximum activity towards acetamide. In contrast acyltransferase B had a broad substrate spectrum with highest acyltransferase activity for nicotinamide and exhibited significant high acyltransferase to amide hydrolase activity ratio for various amides. Purified acyltransferase B was a monomeric protein with molecular mass of 63 kDa. Circular dichroism spectroscopy showed that acyltransferase B was αβ protein with 39% α-helix, 17% β-sheet and 26% random coils. The optimum temperature and pH of acyltransferase B were 45 °C and 7.0 respectively. Catalytic efficiency of acyltransferase B was relatively high with a Kcat/Kmamide and K cat /Km NH 2 OH of 32.0 mM−1 s−1 and 2.47 mM−1 s−1 respectively. Heavy metals and thiol modifying reagents inhibited acyltransferase activity while DTT caused a 2.2 fold increase in activity. Furthermore, acyltransferase B showed tolerance to many polar and non polar organic solvents (10–30%, v/v) which is promising for the synthesis of hydrophobic hydroxamic acids. This acyltransferase B has been shown to be of potential use for hydroxamic acid synthesis owing to its high ratio of acyltransferase to amide hydrolase activities and its considerable organic solvent compatibility.