Characterisation of endoglucanases EGB and EGC from Fibrobacter succinogenes

The enzymatic properties of two endoglucanases from Fibrobacter succinogene s, EGB and EGG, were analysed. EGB and EGC were purified from recombinant E scherichia coli cultures expressing their gene. The failure of purification of EGB by classical techniques led us to produce antipeptide antibodies th at allowed immunopurification of the protein from E. coli as well as its de tection in F. succinogenes cultures. Synthetic peptides were selected from the predicted primary structure of EGB, linked to bovine serum albumin and used as immunogens to obtain specific antibodies. One of the polyclonal ant ipeptide antisera was used to purify EGB. EGC was purified by affinity chro matography with Ni-NTA resin. The endo mode of action of the two enzymes on carboxymethyl-cellulose was different. The values of K-m and V-max were re spectively 13.6 mg/ml and 46 mu mol/min mg protein for EGB, and 7 mg/ml and 110 mu mol/min mg protein for EGG. The reactivity of the antipeptide and t he anti-EGG sera with F. succinogenes proteins of molecular mass different from that of EGB and EGC produced in E. coli suggested post-translational m odification of the two enzymes in F. succinogenes cultures. Expression of e ndB and endC genes in F. succinogenes was confirmed by RT-PCR. (C) 2000 Els evier Science B.V. All rights reserved.