title: Unexpected mode of action of sweet potato β-amylase on maltooligomer substrates.
abstract: β-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme catalyzing the hydrolysis of α(1,4) glycosidic linkages in polysaccharides and removes successively maltose units from the non-reducing ends. The enzyme belongs to glycoside hydrolase GH14 family and inverts the anomeric configuration of the hydrolysis product. Multiple attack or processivity is an important property of polymer active enzymes and there is still limited information about the processivity of carbohydrate active enzymes. Action pattern and kinetic measurements of sweet potato β-amylase were made on a series of aromatic chromophor group-containing substrates (degree of polymerization DP 3-13) using HPLC method. Measured catalytic efficiencies increased with increasing DP of the substrates. Processive cleavage was observed on all substrates except the shortest pentamer. The mean number of steps without dissociation of enzyme-product complex increases with DP of substrate and reached 3.3 in case of CNPG11 indicating that processivity on longer substrates was more significant. A unique transglycosylation was observed on those substrates, which suffer processive cleavage and the substrates were re-built by the enzyme. Our results are the first presentation of a transglycosylation during an inverting glycosidase catalyzed hydrolysis. The yield of transglycosylation was remarkable high as shown in the change of the CNPG11 quantity. The CNPG11 concentration was doubled (from 0.24 to 0.54mM) in the early phase of the reaction.
authors.affiliation: Institute of Inorganic and Analytical Chemistry, Faculty of Sciences and Technology, University of Debrecen, H-4010 Debrecen, P.O. Box 21, Hungary. Electronic address: fazekas.erika@science.unideb.hu.
authors.forename: Erika
authors.initials: E
authors.lastname: Fazekas
authors.forename: Katalin
authors.initials: K
authors.lastname: Szabó
authors.forename: Lili
authors.initials: L
authors.lastname: Kandra
authors.forename: Gyöngyi
authors.initials: G
authors.lastname: Gyémánt
mesh_terms: D055162:Biocatalysis; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002863:Chromogenic Compounds / Q000737:chemistry; D057075:Enzyme Assays; D006031:Glycosylation; D006868:Hydrolysis; D027723:Ipomoea batatas / Q000737:chemistry* / Q000201:enzymology; D007700:Kinetics; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D001614:beta-Amylase / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D002863:Chromogenic Compounds; D010940:Plant Proteins; D008320:Maltose; D001614:beta-Amylase
keywords: HPLC action pattern; Maltooligomers; Processive action; Sweet potato; Transglycosylation; β-Amylase