An automated method for measuring the operational stability of biocatalysts with carbonic anhydrase activity |
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| Institution: | 1. Department of Chemical Engineering, Columbia University, New York, NY 10027, United States;2. Department of Earth and Environmental Engineering, Columbia University, New York, NY 10027, United States;1. Department of Engineering and Applied Sciences, University of Bergamo, Dalmine, Italy;2. Dipartimento di Chimica, Università degli Studi di Milano, Milano, Italy;3. Department of Management, Information and Production Engineering, University of Bergamo, Dalmine, Italy;4. Ricerca sul Sistema Energetico – RSE SpA, Milano, Italy;1. Image Processing Center, School of Astronautics, Beihang University, Beijing 100191, PR China;2. College of Aerospace Science and Engineering, National University of Defense Technology, Changsha 410073, PR China;1. Department of Pharmacy, University of Patras School of Health Sciences, Patras, Greece;2. Department of Pathology, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, Abu Dhabi, United Arabic Emirates;3. Faculty of Medicine and Health Sciences, Department of Pathology, Bioinformatics Unit, Erasmus University Medical Center, Rotterdam, The Netherlands;4. Department of Pathophysiology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece;5. First Department of Dermatology-Venereology, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece;6. General Department, University of Thessaly, Karditsa, Greece;7. The Golden Helix Foundation, London, United Kingdom;1. State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China;2. Suzhou Key Laboratory of Environment and Biosafety, Suzhou 215123, China;3. School of Material Engineering, Jinling Institute of Technology, Nanjing 211169, China |
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| Abstract: | The operational stability of an enzyme can be quantified by its half-life, or the length of time after which 50% of its original activity has degraded. Ideally, continuous methods for measuring half-lives are preferred but they can be expensive and relatively low throughput. Batch methods, while simple, cannot be used for all enzymes. For example, batch reactions can be difficult when there is a gas phase reactant or when there is significant product or substrate inhibition. Here we describe a repeated-batch method for measuring the half-life of carbonic anhydrase (CA)-based biocatalysts by automated periodic switching between a forward and reverse reaction. This method is inexpensive and can be multiplexed for high-throughput analysis of enzyme variants. Several purified CA enzymes as well as whole-cell biocatalysts with engineered CA activity were evaluated with this method. The results indicate a significant increase in operational stability is achieved upon immobilization of CA in the cellular periplasm of Escherichia coli. |
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| Keywords: | Biocatalysis Heterogeneous biocatalysis Immobilized enzymes Enzyme deactivation Carbonic anhydrase Enzyme stability |
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