ABSTRACT
alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 51,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(0)/K(m) value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)-O-alpha-maltosyl-maltose were novel saccharides.
Subject(s)
Acremonium/enzymology , alpha-Glucosidases/chemistry , alpha-Glucosidases/isolation & purification , Carbohydrate Conformation , Disaccharides/metabolism , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Kinetics , Maltose/metabolism , Oligosaccharides/biosynthesis , Protein Subunits , Substrate SpecificityABSTRACT
The gene encoding a glucodextranase from Arthrobacter globiformis I42 was cloned and, subsequently, heterologously expressed in Escherichia coli. This glucodextranase gene consists of 1048 amino acid residues with a calculated molecular mass of 109,135 Da. The roles of two residues at the active site of A. globiformis I42 glucodextranase were examined by site-directed mutagenesis. Glutamic acid residues 458 and 656, which are part of the apparent catalytic residues, were found to be essential for hydrolase activity.
