Regulation and characterization of Thermobifida fusca carbohydrate-binding module proteins E7 and E8.
E7, a single domain Family 33 cellulose binding module (CBM) protein, and E8, a non-catalytic, three-domain protein consisting of a Family 33 CBM, a FNIII domain, followed by a Family 2 CBM, were cloned, expressed, purified, and characterized. Western blots showed that E7 and E8 were induced and secreted when Thermobifida fusca was grown on cellobiose, Solka floc, switchgrass, or alfalfa as well as on beta-1,3 linked glucose molecules such as laminaribiose or pachyman. E8 bound well to alpha- and beta-chitin and bacterial microcrystalline cellulose (BMCC) at all pHs tested. E7 bound strongly to beta-chitin, less well to alpha-chitin and more weakly to BMCC than E8. Filter paper binding assays showed that E7 was 28% bound, E8 was 39% bound, a purified CBM2 binding domain from Cel6B was 88% bound, and only 5% of the Cel5A catalytic domain was bound. A C-terminal 6xHis tag influenced binding of both E7 and E8 to these substrates. Filter paper activity assays showed enhanced activity of T. fusca cellulases when E7 or E8 was present. This effect was observed at very low concentrations of cellulases or at very long times into the reaction and was mainly independent of the type of cellulase and the number of cellulases in the mixture. E8, and to a lesser extent E7, significantly enhanced the activity of Serratia marscescens Chitinase C on beta-chitin.
Department of Molecular Biology and Genetics, Cornell University, 458 Biotechnology Building, Ithaca, New York 14853, USA.
Felix
F
Moser
Diana
D
Irwin
Shaolin
S
Chen
David B
DB
Wilson
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002475:Cellobiose / Q000378:metabolism; D044602:Cellulases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000737:chemistry; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D002688:Chitinases / Q000378:metabolism; D003001:Cloning, Molecular; D003470:Culture Media; D004187:Disaccharides / Q000378:metabolism; D005798:Genes, Bacterial; D005936:Glucans / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D000455:Medicago sativa / Q000378:metabolism; D008969:Molecular Sequence Data; D008897:Panicum / Q000378:metabolism; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003470:Culture Media; D004187:Disaccharides; D005936:Glucans; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins; C014637:laminaribiose; D002686:Chitin; D002475:Cellobiose; D002482:Cellulose; C014731:pachyman; D044602:Cellulases; D002688:Chitinases; C109691:microcrystalline cellulose