Purification, characterization, and subsite affinities of Thermoactinomyces vulgaris R-47 maltooligosaccharide-metabolizing enzyme homologous to glucoamylases.
A maltooligosaccharide-metabolizing enzyme from Thermoactinomyces vulgaris R-47 (TGA) homologous to glucoamylases does not degrade starch efficiently unlike most glucoamylases such as fungal glucoamylases (Uotsu-Tomita et al., Appl. Microbiol. Biotechnol., 56, 465-473 (2001)). In this study, we purified and characterized TGA, and determined the subsite affinities of the enzyme. The optimal pH and temperature of the enzyme are 6.8 and 60 degrees C, respectively. Activity assays with 0.4% substrate showed that TGA was most active against maltotriose, but did not prefer soluble starch. Kinetic analysis using maltooligosaccharides ranging from maltose to maltoheptaose revealed that TGA has high catalytic efficiency for maltotriose and maltose. Based on the kinetics, subsite affinities were determined. The A1+A2 value of this enzyme was highly positive whereas A4-A6 values were negative and little affinity was detected at subsites 3 and 7. Thus, the subsite structure of TGA is different from that of any other GA. The results indicate that TGA is a metabolizing enzyme specific for small maltooligosaccharides.
Ichikawa
Kazuhiro
K
Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Japan.
Tonozuka
Takashi
T
Uotsu-Tomita
Rie
R
Akeboshi
Hiromi
H
Nishikawa
Atsushi
A
Sakano
Yoshiyuki
Y
D000465:Algorithms; D002855:Chromatography, Thin Layer; D004355:Drug Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008320:Maltose / Q000378:metabolism*; D008848:Micromonosporaceae / Q000201:enzymology*; D009844:Oligosaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D009844:Oligosaccharides; D011994:Recombinant Proteins; D008320:Maltose; D005087:Glucan 1,4-alpha-Glucosidase