Enzymatic synthesis of p-nitrophenyl alpha-maltopentaoside in an aqueous-methanol solvent system by maltotetraose-forming amylase: a substrate for human amylase in serum.

Transglycosylation from maltopentaose to the 4-position of p-nitrophenyl alpha-glucoside was efficiently induced through the use of maltotetraose-forming amylase from Pseudomonas stutzeri in an aqueous solution containing methanol at a high concentration. The enzyme specifically formed p-nitrophenyl alpha-maltopentaoside (12% of the enzyme-catalyzed net decrease of maltopentaose) from maltopentaose as a donor and p-nitrophenyl alpha-glucoside as an acceptor. The rate of the transglycosylation depended on the concentration of methanol solvent, the pH and the temperature. Use of the aqueous methanol system in this reaction not only ensured a sufficient solubility of p-nitrophenyl alpha-glucoside but also resulted in a remarkable increase in the formation of p-nitrophenyl alpha-maltopentaoside, which is a useful substrate for assay of human amylase in serum and urine.