title: Catalytic activities of intracellular dimeric neopullulanase on cyclodextrin, acarbose and maltose.
abstract: Multi-substrate specificity of neopullulanase towards cyclodextrin, acarbose and maltose was investigated using a clone originating from Bacillus stearothermophilus IMA6503. The enzyme purified from Escherichia coli harbouring the corresponding nplA gene hydrolysed beta-cyclodextrin (beta-CD) to maltose and glucose. It exhibited substrate preference for beta-CD, starch and pullulan in the proportions of 10.4:1.2:1. The enzyme not only hydrolysed acarbose, an alpha-amylase inhibitor, to a pseudotrisaccharide (PTS) and glucose, but also transferred PTS to glucose, forming isoacarbose. Moreover, it hydrolysed maltose to glucose and transferred the glucose to another maltose molecule to form panose when maltose was present at a low concentration (0.5%) in the reaction solution. The enzyme catalysed condensation between two maltose molecules and subsequent hydrolysis of the resulting 6(2)-O-alpha-maltosyl-maltose to glucose and panose, when maltose concentration was increased to 20%. Neopullulanase was likely to be present in monomer-dimer equilibrium with a molar ratio of 1:9 in 50 mM sodium acetate buffer (pH 6.0). The association-dissociation equilibrium of neopullulanase was shifted to monomerization by KCl. When the content of monomer increased in the reaction mixture, the specific activity towards soluble starch increased to 150%, while that towards beta-CD decreased to 80%. Therefore, multi-substrate specificity of neopullulanase was likely to be modulated by the shift of monomer-dimer association equilibrium.
authors.affiliation: Research Center for New Bio-Materials in Agriculture and Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University, Suwon, 441-744, South Korea.
authors.forename: Kyung-A
authors.initials: KA
authors.lastname: Cheong
authors.forename: Tae-Jip
authors.initials: TJ
authors.lastname: Kim
authors.forename: Jong-Won
authors.initials: JW
authors.lastname: Yoon
authors.forename: Cheon-Seok
authors.initials: CS
authors.lastname: Park
authors.forename: Tae-Soo
authors.initials: TS
authors.lastname: Lee
authors.forename: Young-Bae
authors.initials: YB
authors.lastname: Kim
authors.forename: Kwan-Hwa
authors.initials: KH
authors.lastname: Park
authors.forename: Jung-Wan
authors.initials: JW
authors.lastname: Kim
mesh_terms: D020909:Acarbose / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003505:Cyclodextrins / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000737:chemistry / Q000201:enzymology; D008320:Maltose / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003505:Cyclodextrins; D005936:Glucans; D008320:Maltose; C009109:pullulan; D006026:Glycoside Hydrolases; C058484:neopullulanase; D020909:Acarbose