IDENTIFICATION OF 2 FUNCTIONALLY DIFFERENT CLASSES OF EXOCELLULASES

There are two classes of synergism in cellulase mixtures: synergism be tween endocellulases and exocellulases, and synergism between certain exocellulases. Exocellulases have been defined traditionally as releas ing cellobiose from the nonreducing ends of cellulose, but this defini tion is inadequate to explain exo/exo synergism. Several recent report s indicate that some exocellulases are capable of hydrolyzing cellulos e from the reducing end. The existence of two exocellulase classes wit h different specificities could provide an explanation for exo/exo syn ergism. In this paper, we report the substrate specificity of three Th ermomonospora fusca (E3, E4, and E6) and two Trichoderma reesei (CBH I and CBH II) exocellulases on labeled cellooligosaccharides. We descri be a new nonradioactive technique for determining substrate specificit y, in which ion-spray mass spectrometry was used to analyze the produc ts of enzymatic digests of cellopentaose labeled with O-18 at the redu cing end. Exocellulase reactivity was also investigated on cellopentao se labeled at the nonreducing end with C-14, and cellooligosaccharides reduced with NaBH4. The distribution of label in the reaction product s supports the existence of two functional classes of exocellulases. O ne class (containing CBH I, E4, and E6) preferentially cleaves cellool igosaccharides from the reducing end, while the other (containing E3 a nd CBH II) preferentially cleaves from the nonreducing end. This class ification of exocellulases is consistent with exo/exo synergism experi ments, and with published cellulase crystallographic data.