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.