Effect of substrate concentration and enzyme concentration
Contour plot of both the variables indicates increase in cellulase
activity with increases in both the independent variables and
remains constant to a fixed enzyme to substrate ratio beyond that
increase in substrate concentration inhibits cellulase activity indicating
inhibiting effect of higher substrate concentration (Fig. 9).
For comparison of the analysis using the coded values of the variables,
it is notice that the coefficient for substrate is larger than that
for enzyme concentration (Eq. (4)). This is a scaling effect as the
range for substrate (30–150) was smaller than for enzyme concentration
(200–900). The scaling effect has masked the ‘importance’
effect. When uncoded variables are used the enzyme concentration
coefficient is larger, reflecting that enzyme concentration has also a
larger linear effect. The crude enzymes, endoglucanase and exoglucanase
which act on the surface of an insoluble cellulose substrate
to be synergistic, they must work in close enough proximity to take
advantage of each other’s activity [35]. At lower substrate/enzymes
ratio these exo and endo acting enzyme are relatively close to
one another as compared to higher substrate/enzyme ratio. Thus,
considering the case of randomly associated exo and endo acting
enzymes, synergy will be reduced as substrate concentrations
increase above that required formaximumenzymeadsorption [36].
A high ratio will thus give similarly high surface coverage, leading
to a reduction of the average distance between cellobiohydrolases
and available chain end created by endoglucanase [33]. The loss of
synergy upon surface dilution would be visible as an apparent substrate
inhibition, and such kinetic behavior has been observed with
crude T. reesei cellulase systems [37]. The cellulases have been proposed
to be capable of lateral diffusion on the cellulose surface [38].
This result strongly correlated that maximum activity occurred at
constant cellulase/substrate ratio [33].