Presence of several cellulose-binding proteins in culture supernatant and cell lysate of Eubacterium cellulosolvens 5.

Abstract

Attempts were made to separate and characterize cellulose-binding proteins (CBPs) from both the culture supernatant and cell lysate of Eubacterium cellulosolvens 5. Once the CBPs were bound to Avicel cellulose, they were then effectively eluted with the solution containing 3.2 or 5% sodium dodecyl sulfate (SDS), but not eluted with the solution containing various kinds of carbohydrates and reagents. Namely, CBPs in both the culture supernatant and cell lysate of the bacterium bound tightly and strongly to cellulose. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted CBPs indicated that the CBPs contained the two major proteins having the molecular weights of approximately 160 and 84 kilodaltons (kDa) and one sub-major protein having a molecular weight of approximately 140 kDa. Zymogram analysis after the SDS-PAGE of the eluted CBPs showed that two proteins exhibited the highest levels of carboxymethyl cellulase (CMCase) activity corresponding to the molecular weights of approximately 160 and 90 kDa. A major protein having the molecular weight of approximately 160 kDa exhibited a distinct CMCase activity and was designated as CBPE1. Western immunoblot analysis indicated that the proteins prepared from 16 representative strains of rumen bacteria did not cross-react with rabbit antiserum raised against CBPE1. Thus, CBPE1 may be a unique CBP that plays an important role in the adhesion of the bacterium to cellulose.

Cloning, nucleotide sequence and module structure of the gene encoding the cellulose-binding protein B (CBPB) of Eubacterium cellulosolvens 5

“…E. coli TOP 10 cells (Invitrogen) were used as a host for recombinants made with TOPO TA cloning (Invitrogen) and the pBAD TOPO TA cloning (Invitrogen). Media and culture conditions were described in previous reports (Toyoda and Minato, 2002a, b;Toyoda et al, 2001Toyoda et al, , 2003.…”

“…The binding ability of each recombinant protein to cellulose and cell wall fragments was assayed according to the method described in a previous report (Toyoda et al, 2001), with some modifications. The recombinant protein was partially purified by Ni 2ϩ affinity chromatography from the supernatant of E. coli whole-cell lysate.…”

“…Because adhesion of microbes to cellulose is a prerequisite for the degradation of cellulosic materials (Kudo et al, 1987), mechanisms of bacterial adhesion to cellulose have been investigated (Chesson and Forsberg, 1997;Minato et al, 1993;Miron et al, 2001). It has been found that E. cellulosolvens 5 produces many cellulose-binding proteins (CBPs), and some CBPs show carboxymethylcellulase (CMCase) activity (Toyoda et al, 2001). To begin the study on the adhesion and degradation of cellulose by the organism, we first purified CBPA (the largest protein of CBPs), cloned and sequenced the gene encoding the protein, and clarified the structural features of the protein (Toyoda and Minato, 2002a, b;Toyoda et al, 2001).…”

“…It has been found that E. cellulosolvens 5 produces many cellulose-binding proteins (CBPs), and some CBPs show carboxymethylcellulase (CMCase) activity (Toyoda et al, 2001). To begin the study on the adhesion and degradation of cellulose by the organism, we first purified CBPA (the largest protein of CBPs), cloned and sequenced the gene encoding the protein, and clarified the structural features of the protein (Toyoda and Minato, 2002a, b;Toyoda et al, 2001). The CBPA comprises a catalytic module of glycosyl hydrolase family 9 (GHF9), two linkers, an unknown module (UM-1), a carbohydrate-binding module (CBM), and a cell-wall binding module (CWBM) (Toyoda and Minato, 2002a, b).…”

Biochemical Characterization of Cellulose-Binding Proteins (CBPA and CBPB) from the Rumen Cellulolytic BacteriumEubacterium cellulosolvens5

“…[2][3][4] In previous studies, we identified several proteins bound to crystalline cellulose from E. cellulosolvens 5, 5) and cloned and sequenced the gene encoding CBPA and CBPB. 6,7) CBPA and CBPB comprise the catalytic modules of GHF9, two linkers, family 3 carbohydrate-binding modules (CBM3), novel cellulose-binding modules (CBM), and novel cell-wall binding modules.…”