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Characterization of an Exo‐Maltotetraose‐forming Amylase of Pseudomonas stutzeri and Application in p ‐Nitrophenyl β‐ D ‐Galactosyl‐α‐Maltooligosaccharides Production
Author(s) -
Kubota Shinobu,
Ogawa Koichi,
Nakamura Nobuyuki,
Usui Taich
Publication year - 1997
Publication title -
starch ‐ stärke
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.62
H-Index - 82
eISSN - 1521-379X
pISSN - 0038-9056
DOI - 10.1002/star.19970491108
Subject(s) - pseudomonas stutzeri , chemistry , amylase , hydrolysis , enzyme , molar ratio , stereochemistry , monomer , nuclear chemistry , biochemistry , organic chemistry , catalysis , bacteria , polymer , biology , genetics
Some kinetic characteristics and hydrolytic action patterns on various β‐ D ‐galactosyl‐maltooligosaccharides (Gal‐Gn), ranging in size from D.P. (degree of polymerization) 5 to 8, of an exo‐maltotetraose‐forming amylase of Pseudomonas stutzeri (G4‐amylase) were examined to produce a few p ‐nitrophenyl β‐ D ‐galactosyl‐α‐maltooligosaccharides (Gal‐GnP, n = 4,5). The relative hydrolytic reaction rates for larger Gal‐Gn by the enzyme were larger than those for smaller saccharides tough the values for unmodified linear maltooligosachharides were almost same. Michaelis constants ( K m) for hydrolysis of Gal‐G4, Gal‐G5, Gal‐G6 and Gal‐G7 by the enzyme were 1.3, 1.9, 1.3 and 1.3mM, and apparent molecular activities ( k o) for these saccharides were 5.9, 38, 91 and 126s −1 , respectively. The values of k o/ K m for them were remarkably smaller than those for unmodified linear maltooligosaccharides. The G4‐amylase cleaved 2 points of the α‐1,4‐glucosidic linkage in β‐1,4‐Gal‐G4 to give β‐1,4‐Gal‐G2 and ‐Gal‐G3 in the molar ratio of 3:1, whereas the enzyme attacked 3 points of the linkage in β‐1,4‐Gal‐G5, ‐Gal‐G6 and ‐Gal‐G7 to form β‐1,4‐Gal‐G2,‐Gal‐G3 and Gal‐G4 in the molar ratios of 2:5:1, 1:3:6 and 1:3:6, in the early stage of the reaction, respectively. On the other hand, the enzyme showed no action on β‐1,6‐Gal‐G4 and formed β‐1,6‐Gal‐G4 solely from β‐1,6‐Gal‐G5, and β‐1,6‐Gal‐G4 and ‐Gal‐G5 were from β‐1,6‐Gal‐G6 and ‐Gal‐G7 in the ratios of 8:1 and 2:1, respectively. The enzyme also catalyzed the transfer action to produce Gal‐G3P, Gal‐G4P and Gal‐G5P, of which the formation ratio was coincided well with the hydrolytic action pattern on each Gal‐Gn, from Gal‐Gn tested as a donor and p ‐nitrophenyl α‐glucoside (GP) as an acceptor in an aqueous solution containing 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 assay of human α‐amylase.