Immobilization of β-galactosidase onto Sepharose and stabilization in room temperature ionic liquids

Abstract

The hydrolysis of o-nitrophenyl-β- d-galactopyranoside (ONPG) by β-galactosidase immobilized on Sepharose CL-4B ® was investigated in five different ionic liquids (ILs), 1-butyl-3-methylimidazolium X −; [ X = CF 3SO 3 −, BF 4 −, PF 6 −, CH 3SO 4 −and N(CN) 2 −]. Michaelis–Menten kinetic studies were conducted in phosphate buffer and in the five ionic liquids. For the immobilized enzyme in the ILs, the K m values were lower (0.36–1.2 mmol ONPG) while the V max values were higher (0.04–0.008 min − 1 ) compared to those in aqueous phosphate buffer suggesting a marked increase in the efficiency of the immobilized enzyme in the ionic liquid. For the free enzyme in the ionic liquids, the K m values, in general, were larger (0.45–4.96 mmol ONPG) than those of the immobilized enzyme in the ionic liquid. A postulated mechanism for the hydrolysis is suggested, involving interception of the intermediate oxonium ion species by the counter ion of the ionic liquid, thereby enabling the hydrolysis to occur at a faster rate.