(587n) Study On the Synergism Between Trichoderma Reesei Cellobiohydrolase 1 and Cellobiohydrolase 2 Using Enzyme Cocktail

In past studies, JGC corporation has reported that the decrease in the final yield of the cellulase reaction associated with the reduction in the enzyme loading doesn’t occur under static condition, which we called the phenomenon “static effect” (2010 AIChE Annual meeting). Further investigations revealed that CBH2 was especially vulnerable to agitation, and preservation of CBH2 and its synergism with CBH1 enables the “static effect” (2011 AIChE Annual meeting).  This study investigates further on this synergism between CBH1 and CBH2 that enables the static effect. Experiments were carried out under static condition to avoid CBH2 deactivation. Since the static effect occurs under presence of endoglucanase and β-glucosidase, the investigation used enzyme mixtures containing these enzymes but with only one of the two types of cellobiohydrolase to reflect the reaction condition which “static effect” is exhibited. Mixtures were named CBH1 mixture and CBH2 mixture.

10 w/v% filter paper slurries were hydrolyzed with 2 mg-protein/g-dry substrate of CBH1 mixture or CBH2 mixture alone. Synergism was investigated using the solution which combined 2 mg-protein/g-dry substrate worth of CBH1 mixture and CBH2 mixture, making the total enzyme loading of 4 mg-protein/g-dry substrate. For comparison, hydrolysis using 4 mg-protein/g-dry substrate of CBH1 mixture or CBH2 mixture alone was conducted also.

The combined solution produced more glucose compared to either CBH1 mixture or CBH2 mixture alone under constant enzyme loading. Thus, efficiency of having both cellobiohydrolases in the system is apparent. However, our synergism study using the mixtures didn’t exhibit the synergistic effect that was proven in the past synergism studies using mono-component enzymes. In the past studies, the experimental curve using a combination of CBH1 and CBH2 mono-components was greater than the expected curve calculated by addition of experimental curves using individual enzymes. In our experiment, the experimental curves using 4 mg-protein/g-dry of the combined solution matched the expected curve calculated by adding the glucose concentrations of hydrolysis using 2 mg-protein/g-dry substrate CBH1 mixture and CBH2 mixture. This phenomenon was observed under varying substrate and enzyme concentrations. Moreover, it was not only observed using filter paper, which is a pure cellulosic substrate, but steam exploded eucalyptus also, which is a very heterogeneous substrate.

The reason for this difference between our synergism study using CBH1 mixture and CBH2 mixture compared to the mono-component synergism study in the past is hypothesized to be the absence of restrictions on cellobiohydrolase activity associated with the absence of endoglucanase and β-glucosidase. CBH2 is reported to have endoglucanase activity also, which will enhance the CBH1 activity under absence of endoglucanase. However, the results obtained from the synergism study using the mixtures suggested that under the presence of enough endoglucanases activity, CBH1 and CBH2 work efficiently not by enhancing the activity of each other, but rather, by allowing each other to work at its best capacity without interfering.