title: Characterisation of endoglucanases EGB and EGC from Fibrobacter succinogenes.
abstract: The enzymatic properties of two endoglucanases from Fibrobacter succinogenes, EGB and EGC, were analysed. EGB and EGC were purified from recombinant Escherichia coli cultures expressing their gene. The failure of purification of EGB by classical techniques led us to produce antipeptide antibodies that allowed immunopurification of the protein from E. coli as well as its detection 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 antipeptide antisera was used to purify EGB. EGC was purified by affinity chromatography 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 respectively 13.6 mg/ml and 46 micromol/min mg protein for EGB, and 7 mg/ml and 110 micromol/min mg protein for EGC. The reactivity of the antipeptide and the anti-EGC sera with F. succinogenes proteins of molecular mass different from that of EGB and EGC produced in E. coli suggested post-translational modification of the two enzymes in F. succinogenes cultures. Expression of endB and endC genes in F. succinogenes was confirmed by RT-PCR.
authors.affiliation: Laboratoire de Microbiologie, INRA, Centre de Clermont-Ferrand-Theix, 63122, Saint-Genès-Champanelle, France.
authors.forename: C
authors.initials: C
authors.lastname: Béra-Maillet
authors.forename: V
authors.initials: V
authors.lastname: Broussolle
authors.forename: P
authors.initials: P
authors.lastname: Pristas
authors.forename: J P
authors.initials: JP
authors.lastname: Girardeau
authors.forename: G
authors.initials: G
authors.lastname: Gaudet
authors.forename: E
authors.initials: E
authors.lastname: Forano
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D002417:Cattle; D002480:Cellulase / Q000032:analysis* / Q000378:metabolism; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D011499:Protein Processing, Post-Translational
chemical_list: D001426:Bacterial Proteins; D002480:Cellulase