New substrate specificity of modified porcine pancreatic α-amylase

Abstract Conversion of the substrate specificity of porcine pancreatic α-amylase (PPA) was studied using chemical modification of His residues. Diethyl pyrocarbonate modified His residues in PPA and the activity of the modified PPA for the hydrolysis of the α- d -(1,4) glucoside bond in starch or oligosaccharides decreased to less than 1% of that of the native enzyme. However, the activity for the hydrolysis of the bond between p -nitrophenol and oligosaccharides in p -nitrophenyl oligosaccharides was increased by chemical modification. When the modified PPA was incubated with a proteinaceous α-amylase inhibitor ( M r 60,000) purified from white kidney bean ( Phaseolus vulgaris ), it bound to the inhibitor. As a result, the remaining less than 1% hydrolytic activity of the modified PPA for starch disappeared completely but that for p -nitrophenyl oligosaccharides remained unaltered. The hydrolytic activity of the native PPA for the α- d -(1,4)glucoside bond in oligosaccharides was stronger than that between p -nitrophenyl and oligosaccharides in p -nitrophenyl oligosaccharides. Therefore, when p -nitrophenyl oligosaccharides (three to five glucose residues) were used as substrates for the native PPA, the α- d -(1,4) glucoside bonds in the oligosaccharides were hydrolyzed. However, the modified PPA-inhibitor complex hydrolyzed only the bond between p -nitrophenol and oligosaccharides in p -nitrophenyl oligosaccharides. The above results reveal that, by chemical modification with diethyl pyrocarbonate and biochemical modification with an amylase inhibitor, amylase can be converted to a new exo-type enzyme which hydrolyzes only the bond between p -nitrophenol and oligosaccharides in p -nitrophenyl oligosaccharides.