Cloning,expression and characterization of highly thermo- and pH-stable maltogenic amylase from a thermophilic bacterium Geobacillus caldoxylosilyticus TK4 |
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| Affiliation: | 1. Department of Chemistry, Karadeniz Technical University, Trabzon, Turkey;2. Department of Chemistry, Rize University, Rize, Turkey;3. Department of Biology, Karadeniz Technical University, Trabzon, Turkey;1. Department of Biochemistry, P.D. Patel Institute of Applied Sciences, CHARUSAT, Changa, 388421, Gujarat, India;2. Dr. K.C. Patel Research and Development Center, CHARUSAT, Changa, 388421, Gujarat, India;3. Department of Advance Organic Chemistry, P.D. Patel Institute of Applied Sciences, CHARUSAT, Changa, 388421, Gujarat, India;1. Organic Materials and Fiber Engineering Department and Bionano System Engineering Department, College of Engineering, Chonbuk National University, Jeonju, South Korea;2. Chemical Engineering Department, Faculty of Engineering, Minia University, El Minia, Egypt;3. Chemical Engineering Department, Faculty of Engineering, Jazan University, Jazan, Saudi Arabia;4. Mathematics and Physics Engineering Department, Faculty of Engineering in Matteria, Helwan University, Cairo 11711, Egypt;1. Key Laboratory of Sustainable Development of Polar Fishery, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, China;2. Shanghai Ocean University, College of Food Sciences and Technology, Shanghai, 201306, China;3. Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, China;4. New Hope Liuhe Co. Ltd., Qingdao, 266071, China;5. Jiangsu Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resource, Lianyungang, 222005, China;1. Department of Drug Sciences, University of Catania, Viale Andrea Doria, 6, 95125 Catania, Italy;2. Department of Agricultural and Forest Sciences, University of Palermo, Viale delle Scienze Ed.4, 90128 Palermo, Italy;3. Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano, Italy;4. Consiglio per la Ricerca in Agricoltura e l''analisi dell''economia agraria – Centro di Ricerca per l’Agrumicoltura e le Colture Mediterranee (CRA-ACM), Corso Savoia 190, 95024 Acireale, Italy;5. Department of Soil, Plant and Food Sciences, University of Bari ‘Aldo Moro’, Via Amendola, 165/A, 70126 Bari, Italy;6. Department of Physics and Earth Science, University of Messina, Viale F. Stagno d''Alcontres, 31, Contrada Papardo, 98166 Messina, Italy;7. Department of Agriculture Food and Environment, University of Catania, Via S. Sofia, 98, 95123 Catania, Italy;1. School of Materials Science & Engineering, Nanyang Technological University, Blk N4.1, 50 Nanyang Avenue, Singapore 639798, Singapore;2. Dipartimento di Scienza Applicata e Tecnologia, Politecnico di Torino sede di Alessandria, INSTM Research Unit, Viale Teresa Michel 5, 15100 Alessandria, Italy;1. Graduate Institute of Chinese Medicine, China Medical University, Taichung, Taiwan;2. School of Chinese Medicine, China Medical University, Taichung, Taiwan;3. Department of Medical Research, China Medical University Hospital, Taichung, Taiwan;4. Department of Medical Genetics, Pediatrics and Medical Research, China Medical University Hospital, Taichung, Taiwan;5. Department of Biotechnology, Asia University, Taichung, Taiwan |
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| Abstract: | Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes, belong to glycoside hydrolase family 13. A gene corresponding to MA in Geobacillus caldoxylosilyticus TK4 (GcaTK4MA) was cloned into pET28a(+) vector and expressed in Escherichia coli with 6xHis-tag at the N-terminus. Herein, we report on the biochemical properties of a new thermo- and pH-stable MA. GcaTK4MA has similar properties those of other MAases in terms of the primary structure, preference for CD over starch and having an extra domain at its N- and C-terminals. The recombinant protein was purified efficiently by using one-step nickel affinity chromatography. The purified enzyme exhibited optimal activity for β-CD hydrolysis at 50 °C and pH 7.0. When the enzyme was separately incubated at 4 °C and 50 °C in the buffer solutions (pH 3.0–9.0) up to 7 days, it was seen that the enzyme had the higher stability at 50 °C than 4 °C. The enzyme retained about 80% of its original activity when it was incubated at 50 °C for 7 days. The enzyme activity was significantly inhibited by SDS and EDTA at the final concentration of 1%. These results suggest that this is the first reported MA having an extremely pH- and thermal stabilities. |
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