Is divalent magnesium cation the best cofactor for bacterial -galactosidase?

摘要

-Galactosidase is a metal-activated enzyme, which breaks down the glucosidic bond of lactose and produces glucose and galactose. Among several commercial applications, preparation of lactose-free milk has gained special attention. The present objective is to demonstrate the activity kinetics of -galactosidase purified from a non-pathogenic bacterium Arthrobacter oxydans SB. The enzyme was purified by DEAE-cellulose and Sephadex G-100 column chromatography. The purity of the protein was checked by high-performance liquid chromatography (HPLC). The purified enzyme of molecular weight similar to 95 kDa exhibited specific activity of 137.7 U mg(-1) protein with a purification of 11.22-fold and yield 12.42 . The exact molecular weight (95.7 kDa) of the purified protein was determined by MALDI-TOF. Previously, most of the studies have used Mg+2 as a cofactor of - galactosidase. In this present investigation, we have checked the kinetic behavior of the purified -galactosidase in presence of several bivalent metals. Lowest K-m with highest substrate (ortho-nitrophenyl--galactoside or ONPG) affinity was measured in presence of Ca2+ (42.45 mu M ONPG). However, our results demonstrated that V-max was maximum in presence of Mn+2 (55.98 mu M ONP produced mg(-1) protein min(-1)), followed by Fe+2, Zn+2, Mg+2, Cu+2 and Ca+2. A large number of investigations reported Mg+2 as potential co factor for -galacosidase. However, -galactosidase obtained from Arthrobacter oxydans SB has better activity in the presence of Mn+2 or Fe2+.