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

Abstract

Abstract 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.