title: Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module.
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.
authors: Department of Chemical Technology and Biotechnology, Aalto University, 00076 Aalto, Finland.
authors: Galina
authors: G
authors: Mai-Gisondi
authors: Department of Chemical Technology and Biotechnology, Aalto University, 00076 Aalto, Finland.
authors: Ossi
authors: O
authors: Turunen
authors: Department of Chemical Technology and Biotechnology, Aalto University, 00076 Aalto, Finland.
authors: Ossi
authors: O
authors: Pastinen
authors: Department of Chemical Technology and Biotechnology, Aalto University, 00076 Aalto, Finland.
authors: Nikolaos
authors: N
authors: Pahimanolis
authors: Department of Chemical Technology and Biotechnology, Aalto University, 00076 Aalto, Finland; Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario M5S 3E5, Canada. Electronic address: emma.master@utoronto.ca.
authors: Emma R
authors: ER
authors: Master
mesh_terms: D000115:Acetylesterase / Q000235:genetics / Q000378:metabolism*; D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000031:analogs & derivatives* / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000201:enzymology; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C005062:acetylcellulose; D002482:Cellulose; D000115:Acetylesterase; C081851:acetylxylan esterase
keywords: Acetyl xylan esterase; Carbohydrate binding module; Cellulose acetate; Hemicellulose; Protein engineering