Transglycosylation by barley α-amylase 1 |
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| Institution: | 1. Department of Biochemistry and Molecular Biology, Medical and Health Science Center, Faculty of Medicine, University of Debrecen, H-4012 Debrecen, P.O. Box 6, Hungary;2. Department of Biochemistry, Faculty of Sciences and Technology, University of Debrecen, Debrecen H-4010, Hungary;3. Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark;4. Molecular Enzymology Laboratory, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan;5. Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark;1. Department of Biochemistry, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, 63501, USA;2. Department of Anatomy, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, 63501, USA;3. Department of Microbiology & Immunology, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO, 63501, USA;4. Faculty of Medicine, Cairo University, Cairo, Egypt;1. MTA Alfréd Rényi Institute of Mathematics, Budapest, Hungary;2. Eötvös Loránd University, Budapest, Hungary;3. MTA-ELTE Geometric and Algebraic Combinatorics Research Group, Budapest, Hungary;4. Budapest University of Technology and Economics, Budapest, Hungary;1. MTA Alfréd Rényi Institute of Mathematics, Reáltanoda u. 13-15 Budapest, 1053, Hungary;2. Eötvös Loránd University, Department of Computer Science, Pázmány Péter sétány 1/C Budapest, 1117, Hungary;3. MTA-ELTE Geometric and Algebraic Combinatorics Research Group, Pázmány Péter sétány 1/C Budapest, 1117, Hungary;4. Department of Computer Science and Information Theory, Budapest University of Technology and Economics, 1117 Budapest Magyar tudósok körútja 2, Hungary;1. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191, China;2. Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization, Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, No. 85, Yanshan Road, Yanshan District, Guilin 541006, China;3. Department of Pharmacy, Guangxi Hezhou Municipal People''s Hospital, Hezhou 542899, China;4. Beijing University of Chinese Medicine, Beijing 100102, China;1. USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL, 61604, USA;2. USDA-ARS-Western Regional Research Center, 800 Buchanan Street, Albany, CA, 94710, USA;1. BRD School of Biosciences, Sardar Patel Maidan, Satellite Campus, P. Box No. 39, Sardar Patel University, Vallabh Vidyanagar, 388120 Gujarat, India;2. Sardar Patel Renewable Energy Research Institute, P. Box No. 2, Vallabh Vidyanagar, 388120 Gujarat, India, India |
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| Abstract: | The transglycosylation activity of barley α-amylase 1 (AMY1) and active site AMY1 subsite mutant enzymes was investigated. We report here the transferase ability of the V47A, V47F, V47D and S48Y single mutants and V47K/S48G and V47G/S48D double mutant AMY1 enzymes in which the replaced amino acids play important role in substrate binding at subsites at −3 through −5. Although mutation increases the transglycosylation activity of enzymes, in the presence of acceptors the difference between wild type and mutants is not so significant. Oligomer transfer reactions of AMY1 wild type and its mutants were studied using maltoheptaose and maltopentaose donors and different chromophore containing acceptors. The conditions for the chemoenzymatic synthesis of 4-methylumbelliferyl-α-d-maltooligosaccharides (MU-α-d-MOSs) were optimized using 4-methylumbelliferyl-β-d-glucoside as acceptor and maltoheptaose as donor. 4-Methylumbelliferyl-α-d-maltoside, -maltotrioside, -maltotetraoside and -maltopentaoside have been synthesized. Products were identified by MALDI-TOF MS. 1H and 13C NMR analyses showed that AMY1 V47F preserved the stereo- and regioselectivity. The produced MU-α-d-MOSs of degree of polymerization DP 2, DP 3 and DP 5 were successfully applied to detect activity of Bacillus stearothermophilus maltogenic α-amylase, human salivary α-amylase and Bacillus licheniformis α-amylase, respectively in a fast and simple fluorometric assay. |
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