Expression of rice β-exoglucanase II (OsExoII) in Escherichia coli, purification, and characterization

Enzymes involved in β-glucan breakdown in plants include endoglucanases, exoglucanases and β-glucosidases. Glycoside hydrolase family 3 (GH3) exoglucanases from barley and maize and a few plant GH3 β-glucosidases have been characterized, but none from rice. A few of these enzymes have been expressed in recombinant yeast and plant systems, but bacterial expression of plant GH3 enzymes has not been successful. We expressed the rice GH3 exoglucanase OsExo2 in Escherichia coli as a thioredoxin fusion protein, while other active plant GH3 enzymes could not be produced in this system. The protein was purified over 2000-fold in three chromatographic steps. The enzyme hydrolyzed β-1,3- and β-1,4-linked oligosaccharides and polysaccharides, consistent with a role in cell wall remodeling. Of the oligosaccharides tested, it had highest catalytic efficiency toward laminaritriose, (apparent k_cat/K_m = 37.7 mM^-1s^-1). Among polysaccharides, OsExoII hydrolyzed barley mixed β-glucan and laminarin with similar efficiencies (apparent k_cat/K_m = 3.7 and 3.4 mL mg^-1 s^-1, respectively), but achieved its highest apparent k_cat with lichenan (2.9 s^-1). OsExoII was found to be stimulated by ethylene glycol, which increased the apparent k_cat and decreased the K_m and was transglycosylated. These results imply that E. coli expression may be successful for certain plant GH3 enzymes and OsExoII may be a useful enzyme for application to glycoside production.