Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis.

An endo-beta-1,4-glucanase (CenA) and an exo-beta-1,4-glucanase (Cex) were prepared from Escherichia coli expressing recombinant DNA of the cellulolytic bacterium Cellulomonas fimi. Purification was facilitated by the high affinities of these enzymes for cellulose. An extracellular C. fimi protease cleaved both enzymes in vivo in a highly specific manner. The affinity of the parent enzyme for cellulose was contained independently in an amino-terminal fragment (p20) of CenA and a carboxyl-terminal fragment (p8) of Cex. These fragments contained homologous amino acid sequences which were proposed to comprise cellulose binding domains. Corresponding fragments, p30 from CenA and p35 from Cex, which were unable to bind to cellulose, contained catalytic domains. In both enzymes, the two functional domains were joined by a hinge region consisting solely of prolyl and threonyl residues. The binding domain was excised from CenA by proteolytic cleavage immediately adjacent to the carboxyl terminus of this hinge. Cex was cleaved at an exactly analogous site. p30 and p35 retained several of the catalytic functions of their parent enzymes. However, p30 was less active than intact CenA against microcrystalline cellulose implying a critical role for the binding domain of CenA in the hydrolysis of crystalline substrate.