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, Ct CBM3 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– Ct CBM3 were produced in Pichia pastoris . Ct CBM3 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– Ct CBM3 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 Ct CBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of Ct CBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to Ct CBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.