Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module |
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| Affiliation: | 1. Department of Chemical Technology and Biotechnology, Aalto University, 00076 Aalto, Finland;2. Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada;1. Institute of Infection and Immunity, St George''s, University of London, United Kingdom;2. University of Oxford Centre for Clinical Magnetic Resonance Research, University of Oxford, United Kingdom;3. Department of Preventive Medicine, School of Medicine, Autonoma University of Madrid, Madrid, Spain;4. School of Medicine, University of St Andrews, United Kingdom;5. Institutes of Global Health & Translational Medicine, University of Liverpool, United Kingdom;1. Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea;2. Department of Applied Chemistry and Biological Engineering, Ajou University, Suwon 443-749, Republic of Korea;1. Bio-energy Corporation, Research and Development Laboratory, 2-9-7 Minaminanamatsu, Amagasaki 660-0053, Japan;2. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan;1. Department of Chemistry, College of Natural Science, Sookmyung Women''s University, Seoul 140-742, Republic of Korea;2. School of Civil, Environmental, and Architectural Engineering, Korea University, Seoul 136-713, Republic of Korea;3. Department of Molecular Cell Biology, Samsung Biomedical Research Institute, SungKyunKwan University School of Medicine, Suwon 440-746, Republic of Korea |
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| Abstract: | The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE–CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE–CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates. |
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| Keywords: | Acetyl xylan esterase Cellulose acetate Hemicellulose Carbohydrate binding module Protein engineering |
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