Enzymatic synthesis of p-nitrophenyl alpha-maltoheptaoside by transglycosylation of maltohexaose-forming amylase.

An extracellular maltohexaose-forming amylase [EC 3.2.1.98] from Klebsiella pneumoniae mutant is a normal hydrolytic enzyme that hydrolyzes short-chain amylose (DP = 23) to give about 40% maltohexaose. Transglycosylation from maltoheptaose to the 4-position of p-nitrophenyl alpha-glucoside was efficiently induced through the use of maltohexaose-forming amylase in an aqueous methanol solution. The enzyme specifically produced p-nitrophenyl alpha-maltoheptaoside (13% of the p-nitrophenyl alpha-glucoside) from maltoheptaose as a donor and p-nitrophenyl alpha-glucoside as an acceptor. The yield of p-nitrophenyl alpha-maltoheptaoside depended on the concentration of methanol solvent, the pH, and temperature. Furthermore, the use of the aqueous methanol system in the reaction not only improved the solubility of p-nitrophenyl alpha-glucoside but also greatly increased the formation of p-nitrophenyl alpha-maltoheptaoside, which is a useful substrate for assay of human amylase in serum and urine.