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

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 = CF3SO3−, BF4−, PF6−, CH3SO4−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 Km values were lower (0.36-1.2 mmol ONPG) while the Vmax 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 Km 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.