MALTOSE PHOSPHORYLASE FROM LACTOBACILLUS-BREVIS - PURIFICATION, CHARACTERIZATION, AND APPLICATION IN A BIOSENSOR FOR ORTHO-PHOSPHATE

With the goal to obtain maltose phosphorylase as a tool to determine o rtho-phosphate, the enzyme from Lactobacillus brevis was purified to 9 8% by an expeditious FPLC-aided procedure which included anion exchang e chromatography, gel filtration, and hydroxyapatite chromatography. T he native maltose phosphorylase had a molecular mass of 196 kDa and co nsisted of two 88 kDa subunits. In isoelectric focusing two isoforms w ith pI values of 4.2 and 4.6 were observed. Maximum enzyme activity wa s obtained at 36 degrees C and pH 6.5 and was independent of pyridoxal 5'-phosphate. The apparent K-m values with maltose and phosphate as s ubstrates were 0.9 mmol l(-1) and 1.8 mmol l(-1), respectively. Maltos e phosphorylase could be stored in 10 nM phosphate buffer pH 6.5 at 4 degrees C with a loss of activity of only 7% up to 6 months. The stabi lity of the enzyme at high temperatures was enhanced significantly usi ng additives like phosphate, citrate, and imidazole. The purified malt ose phosphorylase was used as key enzyme in a phosphate sensor consist ing of maltose phosphorylase and glucose oxidase. A detection limit of 0.1 mu M phosphate was observed and the sensor response was linear in the range between 0.5 and 10 mu M. (C) 1997 Elsevier Science Inc.