Introduction
Thermobifida fusca YX is an aerobic, moderately thermophilic cellulolytic bacterium that produces and secretes a large number of proteins when it is grown on cellulose as a sole carbon source (Gomez del Pulgar and Saadeddin, 2014).  These include six cellulases (Cel5A, Cel6A, Cel6B, Cel9A, Cel9B, and Cel48A), two AA10 proteins, several hemicellulases as well as a number of uncharacterized proteins (Jeoh et al., 2002, Irwin et al., 2003). All of these proteins have had their genes cloned and have been expressed in E.coli , purified, and characterized (Jung et al., 2002, Wilson, 2004, Posta et al, 2004).
One cellulase, Cel9B, was determined to be a β-1,4-endocellulase due to its ability to reduce the viscosity of a carboxymethyl cellulose (CMC) solution over time (Calza et al., 1985, Irwin et al., 1993). Cel9B has a complex domain structure conformed by a N-terminal family 4 carbohydrate binding module (CBM) linked to the protein catalytic domain, which is linked to a fibronectin-like (Fn3) domain, followed by an Eset domain (an Ig-like domain that may play a role in enzyme folding) and ending with a carbohydrate binding module family 2 (CBM2) (Kataeva et al., 2002, 2005, Posta et al., 2004). This enzyme has extremely high activity on CMC but very low activity on crystalline cellulose. This domain structure is very similar to the structure presented onClostridium thermocellum cellulosomal cellulase, CBHA, that shows quite different activity compared to Cel9B (Alahuhta et al., 2010). CBHA was originally thought to be an exocellulase (Schubot et al., 2004). However, its unliganded crystal structure shows an open active site cleft that is characteristic of endocellulases (Kataeva et al., 2005). The endocellulase nature of CBHA was confirmed by several definitive assays (Kataeva et al., 2004).  Although Cel9B has been cloned and its protein product purified, the role of the functional domains in the hydrolysis of biomass substrates have not been resolved.
In this paper, we have cloned, expressed, and characterized a number of Cel9B constructs, in which one or more domains were deleted in order to try to understand the roles of the individual domains in activity and substrate binding on a variety of substrates: Bacterial microcrystalline cellulose (BMCC), phosphoric acid-swollen cellulose (PASC), α-chitin, β-chitin, xylan (birchwood), and xyloglucan. The study offers insight as to how these functional domains might interact with each other to assist in the process of breaking down recalcitrant cellulose.