Discussion
The vast majority of microbial cellulases and other cellulose- degrading
enzymes such as xylanases are organized into a modular format which can
be divided into catalytic and non-catalytic domains. One or a few
carbohydrate - binding motifs (CBMs), responsible for binding to
different substrates, are associated with the catalytic domain (CD) of
many microbial cellulases. Each cellulase possesses its own unique
assortment of CBMs. CBMs are often separated from other cellulase
modules by linker molecules, which are believed to assist in the process
of targeting the CD to different types of substrate (Boraston et al.,
1998).
Cel9B of T. fusca follows this modular organization by
including both CBM2 and CBM4, which are separated from the CD by a
fibronectin-like domain (Fn3) and an Eset domain, respectively. The
arrangement of the catalytic domain followed by a CBM2 that is found in
Cel9B is also common in a number of enzymes produced by bacteria as well
as other organisms, suggesting a common gene transfer event may have
taken place in the distant past (Gao et al., 2010, Li et al., 2009).
Experiments carried out in this paper using truncation mutants and
different substrates have indicated that mutants keeping the CBM2 domain
bind substrates at higher yield that the other mutants, and increase
cellulase activity against both crystalline and amorphous cellulose.
This was clearly demonstrated using wild type Cel9B as well as
truncation mutant M2, which lacks the CBM4. These results agree with for
the work performed for other cellulases which exhibit higher levels of
activity in the presence of CBM2, including Pseudomonas ,Cellulomonas fini , and Cellvibrio japonicus (Boraston et
al., 1998, Tomme et al., 1996, Gilbert et al., 2013, Attia et al, 2016).
CBM2 is one of the most prevalent carbohydrate binding modules and is
over- represented in thermophilic microorganisms. It is possible that
under thermophilic conditions, CBM2 binding to cellulose may be more
stable than those of other CBMs (Moser et al., 2008). Xylan-specific
binding of CBM2 has been demonstrated in the past by Simpson et al.,
(2000). The fact that CBM2 can bind to both amorphous as well as
crystalline cellulose could imply that there are differences in CBM2
function on these vastly different substrates. It has been suggested
that CBM2s may enhance cellulose activity through its binding mechanism,
either by bringing the catalytic domain proximal to the substrate itself
or by disrupting and displacing recalcitrant substrate so that more of
it is made to be accessible to the catalytic domain (Knowles et al.,
1988; Din et al., 1991, Tomme et al., 1996).
Truncation mutant ΔFn3, which lacks an intact Fn3 domain, displayed
reduced substrate binding and cellulase activity. It is possible that
this protein is misfolded. During the production strain grew slowly
(approximately 8 hours to reach OD 0.6 rather than 3-4 hours for the
others) and the protein bound poorly to the nickel column. Unlike the
other truncation mutant proteins, all of Fn3 came out in the flowthrough
and did not bind to the nickel column, as determined by activity assay
(Table 2). Brunecky et al., 2012, suggested that the Fn3 domain aids in
the thermostability of the protein and may in fact function as a spacer
between other functional domains. It is possible, therefore, that the
Fn3 domain may have some indirect stabilizing role in the protein that
was disrupted upon deletion, or perhaps it aids in the folding of the
native protein structure. Fn3 domains have also been thought to play
roles in regulating CBM interactions with substrates (Kataeva et al.,
2002, Cadena et al., 2010). Although removal of the Fn3 domain in the
ΔFn3 mutant appears to have impeded substrate binding, as shown in Table
3, it is more likely that this is really the result of improper protein
folding, as truncation mutant Fn3 (containing solely the catalytic
domain and the Fn3 domain) does not bind to substrate. Other research
groups have shown that Fn3 domain does not affect enzyme binding, rather
it can disrupt the surface of substrate so that single cellulase chains
are directed to the catalytic site, and thus play a role in regulating
catalysis on specific substrates (Kataeva et al., 2002, Guillen et al.,
2010).
Truncation mutants M1 and ΔE9B, containing CBM4, exhibited pronounced
activity on xyloglucan and BMCC that was missing from the other
truncation mutants. Truncation mutant ΔE9B lacks the Eset domain, an
Ig-like domain that may play a role in enzyme folding (Kataeva et al.,
2005). This Eset domain is retained in the M1 mutant (Figure 1). CBM4 is
known to bind to crystalline cellulose as well as amorphous cellulose
(Alahuhta et al., 2010). In this study, truncation mutant M1 exhibited
binding to BMCC, albeit at almost half of the yield of the M2 mutant
that contains the CBM2 domain. Truncation mutant ΔE9B was not able to
bind strongly to BMCC (5% binding yield) yet exhibited activity on this
substrate. Furthermore, both M1 and ΔE9B showed no ability to bind to
xyloglucan (data not shown) yet displayed significant activity on this
substrate. These results bring into question the role of CBM4 in Cel9B.
The relatively high activity of these two mutants on xyloglucan (with
respect to the wild type) and their poor ability to bind to substrates
would suggest that CBM4 plays a role that differs in general from CBM2.
Blake et al., (2006) have demonstrated that CBM4 binding to cellulose
chains is limited but requires access to the entire width of a cellulose
chain. Indeed, this different binding of CBM4 may be necessary for
Cel9B to efficiently catalyze areas of the cell wall that cannot be
targeted effectively by CBM2. The poor binding ability to various
substrates may indicate that CBM4 plays a role at a later stage of cell
wall degradation, where substrates that are available to interact with
CBM4 differ from those that are found early on in the degradation
process.
Truncation mutants cd9B, E9B, and Fn3 contain the catalytic domain and
no CBMs. These mutants exhibited very low yield binding (below 10%) to
any of the substrates tested in this study. While all were able to
display catalytic activity, cd9B displayed the most, suggesting that
retention of the Eset domain and fibronectin-like domain (in E9B and Fn3
mutants, respectively) may have a deleterious effect on the folding of
cd9B that impacts its catalytic activity.
WT, M1, and M2 have the highest and similar activities on PASC while the
other mutants have activities which are similar to each other, but
somewhat lower than WT. This shows that either CBM can improve activity
on this substrate. PASC is an amorphous insoluble substrate with much
higher surface area than BMCC. On the other hand, the activity results
on BMCC show that deleting CBM4 (M1 and ΔE9B) causes a large increase in
activity for M1 and a small increase for ΔE9B over time with respect of
the wild type. This suggests that for Cel9B, CBM4 is not important for
crystalline cellulose activity. Furthermore, deleting the Eset domain
decreases the activity of M1 for reasons that would require further
research.
Since CMC is a soluble substrate, removing the CBMs would not be
expected to affect this activity, and the removal of CBMs presents
increased catalytic activity on CMC for several other cellulases. The
activities on PASC and xylan are very similar to the activities on CMC
and this may be because both are partially soluble. In addition, these
two substrates are not as highly ordered as cellulose, so that binding
domains may not be required for their hydrolysis.