CHARACTERIZATION OF AN EXO-MALTOTETRAOSE-FORMING AMYLASE OF PSEUDOMONAS-STUTZERI AND APPLICATION IN P-NITROPHENYL BETA-D-GALACTOSYL-ALPHA-MALTOOLIGOSACCHARIDES PRODUCTION

Some kinetic characteristics and hydrolytic action patterns on various beta-D-galactosyl-maltooligosaccharides (Gal-Gn), ranging In size. fr om D.P. (degree of polymerization) 5 to 8, of an exo-maltotetraose-for ming amylase of Pseudomonas stutzeri (G4-amylase) were examined to pro duce a few p-nitrophenyl beta-D-galactosyl-alpha-maltooligosaccharides (Gal-GnP, n=4,5). The relative hydrolytic reaction rates for larger G al-Gn by the enzyme were larger than those for smaller saccharides tou gh the values for unmodified linear maltooligosachharides were almost same. Michaelis constants (Km) for hydrolysis of Gal-G4, Gal-G5, Gal-G 6 and Gal-G7 by the enzyme were 1.3, 1.9, 1.3 and 1.3 mM, and apparent molecular activities (ko) for these saccharides were 5.9, 38, 91 and 126s(-1), respectively, The values of ko/Km for them were remarkably s maller than those for unmodified linear maltooligosaccharides. The G4- amylase cleaved 2 points of the alpha-1,4-glucosidic linkage in beta-1 ,4-Gal-G4 to give beta-1,4-Gal-G2 and -Gal-G3 in the molar ratio of 3: 1, whereas the enzyme attacked 3 points of the linkage in beta-1,4-Gal -G5, -Gal-G6 and -Gal-G7 to form beta-1,4-Gal-G2. -Gal-G3 and Gal-G4 i n the molar ratios of 2:5:1, 1:3:6 and 1:3:6, in the early stage of th e reaction, respectively. On the other hand, the enzyme showed no acti on on beta-1,6-Gal-G4 and formed beta-1,6-Gal-G4 solely from beta-1,6- Gal-G5, and beta-1,6-Gal-G4 and -Gal-G5 were from beta-1,6-Gal-G6 and -Gal-G7 in the ratios af 8:1 and 2:1, respectively. The enzyme also ca talyzed the transfer action to produce Gal-G3P, Gal-G4P and Gal-G5P, o f which the formation ratio was coincided well with the hydrolytic act ion pattern an each Gal-Gn, from Gal-Gn tested as a donor and p-nitrop henyl alpha-glucoside (GP) as an acceptor in an aqueous solution conta ining 40% (v/v) methanol. By using this novel reaction, Gal-G5P is now producing on an industrial scale to apply as a substrate for the assa y of human alpha-amylase.