Site-saturation mutagenesis of proline 176 in Cyclodextrin Glucosyltransferase from Bacillus sp. Y112 effects product specificity and enzymatic properties |
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| Institution: | 1. Key Laboratory of Sustainable Development of Polar Fishery, Ministry of Agriculture and Rural A? ;airs, 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. Jiangsu Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resource, Lianyungang, 222005, China;1. Microbial Biotechnology and Bioprocess Engineering (MBBE) Group, Department of Microbiology, Faculty of Science, University of Maragheh, Maragheh, Iran;2. Department of Biological Science, Faculty of Science, University of Kurdistan, Sanandaj, Iran;3. Department of Microbiology, Faculty of Biological Science, Alzahra University, Tehran, Iran;1. Laboratory of Bioprocess Technology, Department of Microbiology, Maharshi Dayanand University, Rohtak, 124001, Haryana, India;2. Department of Botany, Pt. N.R.S. Govt College, Rohtak, 124001, Haryana, India;3. Department of Biotechnology, Central University of Haryana, Jant-Pali, Mahendergarh, 123031, Haryana, India;1. Industrial Bio-materials Research Center, KRIBB, Daejeon 34141, Republic of Korea;2. Department of Biological Sciences, Andong National University, Andong 36729, Republic of Korea;3. Insect Biotech Co. Ltd., Daejeon 34054, Republic of Korea;1. State Key Laboratory of Biochemical Engineering, Beijing Key Laboratory of Biomass Refining Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China;2. University of Chinese Academy of Sciences, Beijing 100049, PR China;1. School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso (PUCV), Valparaíso, Chile;2. Department of Biology, Faculty of Chemistry and Biology, Universidad de Santiago de Chile (USACH), Santiago, Chile;1. School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China;2. Department of Agricultural and Biosystems Engineering, Faculty of Agriculture, Benha University, P.O. Box 13736, Moshtohor, Qaluobia, Egypt;3. Faculty of Agriculture, University for Development Studies, Tamale, Ghana |
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| Abstract: | Based on the analysis of amino acid sequence and simulated structure, saturation mutagenesis was performed to explore the role of the site p176 of cyclodextrin glucosytransferase (CGTase) from Bacillus sp. Y112. Compared to the wild-type, mutant P176G showed 10.4 % improvement in conversion from starch to cyclodextrins (CDs), whose β-CD yield increased by 6% and α-CD yield decreased by 8%. Mutants P176L and P176I were increased by 7.9 % and 9.4 % on CDs production, indicating replacement of hydrophobic amino acids significantly improved in cyclization activity. Kinetics studies indicated the substrate af?nity of P176G and P176K were increase by 13 % and 14 %, and the catalytic ef?ciency of P176K was increase by 14 %. In addition, the optimal temperature of mutants transformed from 50℃ to 40℃ and the optimal pH shifted from 10.0 to 8.0. These results indicate that the site P176 plays a critical role in catalytic activity, product specificity and enzymatic properties of CGTase. |
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| Keywords: | Cyclodextrin glycosyltransferases Site-saturation mutagenesis Site Pro176 Cyclodextrin Product speci?city |
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