INHIBITION OF THE EXO-BETA-1,4-GLUCANASE FROM RUMINOCOCCUS-FLAVEFACIENS FD-1 BY A SPECIFIC MONOCLONAL-ANTIBODY

A monoclonal antibody (MAbS1) specific for exo-beta-1,4-glucanase A (E xoA) from Ruminococcus flavefaciens FD-1 was generated from homogeneou s enzyme. Immunoblots showed that MAbS1 reacted strongly with the ExoA polypeptide and weakly with other polypeptides in the crude cellulase preparation from this organism. However, immunoblots of V8 protease-d igested crude cellulase preparations suggest that MAbS1 also recognize s proteins in R. flavefaciens FD-1 other than ExoA, or that ExoA exist s in multiple forms. Immunoprecipitations using MAbS1 and crude cellul ase preparations specifically removed p-nitrophenyl-beta-D-cellobiosid ase (PNPCase) activity, with little removal of carboxymethylcellulase or C-14-cellulase activities. Immunoprecipitation pellets contained th e ExoA polypeptide as well as two lower-molecular-weight polypeptides which were present in much lower concentrations. The antibody is also suitably specific towards ExoA for use in affinity purification ofthe exoglucanase from culture supernatants of R. flavefaciens FD-1. MabS1 inhibits PNPCase (exoglucanase) activity in vitro. Moreover, the antib ody when added to growing cultures of R. flavefaciens FD-1 decreased t he rate and extent of cellulose hydrolysis. These data suggest that ex oglucanase A is an important component ofthe R. flavefaciens FD-1 cell ulase system. The results show that a specific monoclonal antibody dir ected rewards ExoA inhibits the catalytic activity of this enzyme and decreases the rate and extent of cellulolysis by this microorganism. T hus, ExoA is a vital component of the cellulase system of R. flavefaci ens FD-1, and its activity may be a rate-limiting step in cellulolysis .