Kc. Doerner et al., INHIBITION OF THE EXO-BETA-1,4-GLUCANASE FROM RUMINOCOCCUS-FLAVEFACIENS FD-1 BY A SPECIFIC MONOCLONAL-ANTIBODY, Enzyme and microbial technology, 16(1), 1994, pp. 2-9
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
.