Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents |
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| Institution: | 1. Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan;2. Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan;1. Instituto Superior de Agronomia, Universidade de Lisboa, LEAF, Lisbon, Portugal;2. Departament d’Enginyeria Quimica, Biològica i Ambiental (EE), Universitat Autònoma de Barcelona, Barcelona, Spain;3. Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), Guadalajara, Jalisco, Mexico;1. Department of Chemistry and Biology, Graduate School of Science and Engineering, Ehime University, Matsuyama, Japan;2. Faculty of Science, Ehime University, Matsuyama, Japan;1. State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China;2. School of Biology and Biological Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Panyu, Guangzhou 510006, China;3. College of Life and Geographic Sciences, Kashgar University, Kashgar 844000, China;4. The Key Laboratory of Ecology and Biological Resources in Yarkand Oasis at Colleges & Universities Under the Department of Education of Xinjiang Uygur Autonomous Region, Kashgar University, Kashgar 844000, China;1. Departamento de Biocatálisis, ICP-CSIC, Campus UAM-CSIC, Madrid, Spain;2. Food Biotechnology Division, Biotechnology Research Center (CRBt), Constantine, Algeria;3. Faculty of Nature and life Sciences, Ibn Khalboun University, Tiaret, Algeria;4. Heterogenous Biocatalysis laboratory, CIC Biomagune, Paseo Miramon 182, 20014, San Sebastian, Spain;5. IKERBASQUE, Basque Foundation for Science, Bilbao, Spain;1. School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832003, Xinjiang, PR China;2. School of Life Science and Technology, Beijing Institute of Technology, Beijing, 100081, PR China |
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| Abstract: | Silica-based immobilized metal affinity chromatography adsorbents with various ligand densities were prepared for the purification and immobilization of poly(His)-tagged d-hydantoinase (DHTase). An adsorbent with a ligand density of 13.0 μmol Cu2+/g gel exhibiting the optimal selectivity and a capacity of 1.4 mg/g gel toward the poly(His)-tagged enzyme was identified. The adsorbent was used for the one-step purification of His-tagged enzymes from crude cell lysate with a purity above 90%. The silica-based affinity adsorbents are particularly well suited for industrial scale operations due to their robustness. A packed-bed bioreactor with the DHTase-loaded adsorbents was used for the continuous conversion of d,l-p-hydroxyphenylhydantoin (d,l-HPH) to N-carbamoyl-d-hydroxyphenylglycine, an intermediate for the production of d-hydroxylphenylglycine. Under optimal conditions, 60 °C and pH 8.0, a conversion of 60% was obtained at a residence time of 30 min. Upon extended operation, the catalytic activity of the biocatalysts declined significantly due to enzyme leakage and enzyme denaturation. The extent of enzyme leakage can be attenuated by crosslinking with glutaraldehyde. In this study, we successfully demonstrate that a packed-bed bioreactor containing silica-based IMAC adsorbents can be used for the direct purification and immobilization of poly(His)-tagged enzymes for biotransformation. |
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