Regulation and characterization of Thermobifida fusca carbohydrate-binding module proteins E7 and E8

• Published in: Biotechnology and bioengineering 2008-08, Vol.100 (6), p.1066-1077
• Main Authors: Moser, Felix, Irwin, Diana, Chen, Shaolin, Wilson, David.B
• Format: Article
• Language: English
• Subjects: Actinomycetales - enzymology; Actinomycetales - genetics; Amino Acid Sequence; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Biological and medical sciences; Biotechnology; carbohydrate-binding module (CBM); Carbohydrates; CBM family 33; Cellobiose - metabolism; cellulase; Cellulases - chemistry; Cellulases - genetics; Cellulases - isolation & purification; Cellulases - metabolism; Cellulose; Cellulose - chemistry; CelR; Chitin - chemistry; Chitin - metabolism; chitinase; Chitinases - metabolism; Cloning, Molecular; Culture Media; Disaccharides - metabolism; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Glucans - metabolism; Hydrogen-Ion Concentration; Kinetics; MALDI and ESI mass spectrometry; Mass spectrometry; Medicago sativa - metabolism; Molecular Sequence Data; Panicum - metabolism; Protein Binding; Proteins; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - genetics; Receptors, Cell Surface - isolation & purification; Receptors, Cell Surface - metabolism; Sequence Alignment; Serratia; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Thermobifida fusca
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.21856

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 β-1,3 linked glucose molecules such as laminaribiose or pachyman. E8 bound well to α- and β-chitin and bacterial microcrystalline cellulose (BMCC) at all pHs tested. E7 bound strongly to β-chitin, less well to α-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 β-chitin. Biotechnol. Biotechnol. Bioeng. 2008;100: 1066-1077.