Active-site mutations improved the transglycosylation activity of Stenotrophomonas maltophilia chitinase A |
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| Institution: | 1. Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China;2. China Leather and Footwear Industry Research Institute (Jinjiang) Co., Ltd, Fujian 362261, China;3. Biotechnology Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China;1. Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing, China;2. College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing, China;1. Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, People’s Republic of China;2. College of Life Sciences, Yunnan Normal University, Kunming 650500, People’s Republic of China;3. Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming 650500, People’s Republic of China;4. Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming 650500, People’s Republic of China;1. College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China;2. Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China;3. Istituto di Chimica del Riconoscimento Molecolare, CNR, v. Mario Bianco 9, Milan 20131, Italy;1. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China;2. Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing 100083, China |
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| Abstract: | Transglycosylation (TG) by family 18 chitinases is of special interest due to the many biological applications of long-chain chitooligosaccharides (CHOS). In the current study, the TG activity of chitinase A from Stenotrophomonas maltophilia (StmChiA) was improved through structure-guided mutations within and around the active site. Three independent mutants were created, targeting Trp residues from the ? 3 and ? 1 subsites and the central catalytic Asp from the DxDxE motif of StmChiA. The former was replaced with Ala and the latter with Asn. Changes in the hydrolytic and TG activities of the enzymes were assessed by monitoring the product profile of each mutant by high-performance liquid chromatography. All three mutants showed increased TG activity. Increased in the higher TG activity of mutant W306A was accompanied by increased hydrolysis. However, this mutant also accumulated substantial amounts of TG products during the first 15–30 min of the reaction. In contrast, mutants D464N and W679A showed reduced hydrolysis, which was accompanied by the gradual accumulation of TG products up to 12 h. Molecular docking studies with chitohexaose showed that the side chains of Trp residues mediate stacking interactions with sugar residues from the ? 3 and ? 1 subsites, indicating the importance of these residues in the enzymatic activity of StmChiA. Overall, mutants of the glycon-binding site (W306A and W679A) appear to produce long-chain CHOS more efficiently than the catalytic mutant D464N. |
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