title: A continuous coupled spectrophotometric assay for debranching enzyme activity using reducing end-specific α-glucosidase.
abstract: A novel continuous spectrophotometric assay to measure the activity of the debranching enzyme and α-amylase has been developed. The assay mixture comprises the debranching enzyme (GlgX from Escherichia coli) or α-amylase (PPA from porcine pancreas), a reducing end-specific α-glucosidase (MalZ), maltodextrin-branched β-cyclodextrin (Glcn-β-CD) as the substrate, and the glucose oxidase/peroxidase system (GOPOD). Due to its high reducing end specificity, the branch chains of the substrates are not hydrolyzed by MalZ. After hydrolysis by GlgX or PPA, the released maltodextrins are immediately hydrolyzed into glucose from the reducing end by MalZ, whose concentration is continuously measured by GOPOD at 510 nm in a thermostat spectrophotometer. The kinetic constants determined for GlgX (Km = 0.66 ± 0.02 mM and kcat = 76.7 ± 1.5 s(-1)) are within a reasonable range compared with those measured using high-performance anion-exchange chromatography (HPAEC). The assay procedure is convenient and sensitive, and it requires lower concentrations of enzymes and substrate compared with dinitrosalicylic acid (DNS) and HPAEC analysis.
authors.affiliation: Department of Foodservice Management and Nutrition, Sangmyung University, Seoul 110-743, Republic of Korea.
authors.forename: Viet Ha
authors.initials: VH
authors.lastname: Do
authors.affiliation: Department of Foodservice Management and Nutrition, Sangmyung University, Seoul 110-743, Republic of Korea; Department of Food Technology, An Giang University, An Giang 076, Viet Nam.
authors.forename: Phuong Lan
authors.initials: PL
authors.lastname: Tran
authors.affiliation: Institute of Food Science and Technology, Fuzhou University, Fuzhou, Fujian 350108, China.
authors.forename: Li
authors.initials: L
authors.lastname: Ni
authors.affiliation: Department of Foodservice Management and Nutrition, Sangmyung University, Seoul 110-743, Republic of Korea; School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Republic of Korea. Electronic address: parkkh@snu.ac.kr.
authors.forename: Kwan Hwa
authors.initials: KH
authors.lastname: Park
mesh_terms: D002852:Chromatography, Ion Exchange; D057075:Enzyme Assays / Q000379:methods*; D005964:Glucosyltransferases / Q000378:metabolism; D006004:Glycogen Debranching Enzyme System / Q000378:metabolism*; D007517:Isoamylase / Q000378:metabolism; D007700:Kinetics; D011134:Polysaccharides / Q000737:chemistry; D011549:Pseudomonas / Q000201:enzymology; D020156:Salicylic Acid / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology; D000520:alpha-Glucosidases / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000032:analysis / Q000378:metabolism
chemical_list: D006004:Glycogen Debranching Enzyme System; D011134:Polysaccharides; D047392:beta-Cyclodextrins; C008315:maltodextrin; D005964:Glucosyltransferases; C436710:maltosyltransferase; D000520:alpha-Glucosidases; D007517:Isoamylase; C031215:betadex; D020156:Salicylic Acid
keywords: Continuous coupled assay; Debranching enzyme; Glucose oxidase/peroxidase (GOPOD); Maltodextrin glucosidase (MalZ); Maltodextrin-branched β-cyclodextrin (Glc(n)-β-CD)