263409 Mechanism of Exo-Exo Synergism Between CBH1 and CBH2 During the Hydrolysis of Cellulose

Tuesday, October 30, 2012: 1:20 PM
334 (Convention Center )
Makoto Ikeo1, Shohei Okino1, Yoshiki Ueno2 and Daisuke Taneda1, (1)Technology Development Department, JGC Corporation, Higashiibaraki-gun, Ibaraki Prefecture, Japan, (2)Technology Innovation Center, JGC Corporation, Higashiibaraki-gun, Ibaraki Prefecture, Japan

In past studies, JGC corporation has reported that the efficiency of enzymatic hydrolysis of cellulose using cellulase with low enzyme loadings can be significantly improved by placing the reaction under static condition (2010 AIChE Annual Meeting, 617c Breaking the Barrier to Obtain High Yields On Enzymatic Hydrolysis Using Low Enzyme Loadings). The hydrolysis of 100ml 10% filter paper slurry at the enzyme loading of 6 mg protein/g-dry substrate was conducted using Cellulase SS (Nagase Chemtex). The agitated condition obtained a hydrolyzate with a final glucose concentration of 60 g/L. In contrast, a hydrolyzate with a final glucose concentration of 90 g/L was obtained under the static condition. 

Further investigations found that this improvement occurs by the preservation of CBH2 and the exo-exo synergism between it and CBH1 under the static condition. Cellulase SS solution was agitated without any substrates under 50 °C, and SDS-PAGE analysis was conducted to measure the concentration of individual components. Of all the cellulase components, only the band of CBH2 disappeared within 6 hours of agitation, indicating its deactivation. Further examination using fractionated enzyme solutions, one of which lacked CBH1 and the other lacking CBH2, revealed that both CBH1 and CBH2 are required for the efficient hydrolysis of cellulose.

In this study, the mechanism of the exo-exo synergism between CBH1 and CBH2 was investigated by comparing the characteristics of enzyme adsorption and desorption to the cellulose under agitated and static conditions. The hydrolysis of 10% filter paper slurry with the enzyme loading of 12 mg-protein/ g-dry-substrate under both static and agitated conditions were conducted, and the changes in concentration of each cellulase component in the hydrolyzate was monitored using SDS-PAGE and a densitograph. The glucose concentrations of the hydrolyzates obtained were 88 g/L and 60 g/L under the static and agitated conditions respectively. Under both conditions, the CBH1 and CBH2 concentrations in the hydrolyzate dropped sharply, showing concentration decreases of 100% and 80% respectively, at the initial stage of the reaction, indicating their adsorption to the substrate. Under the static condition in which CBH2 is active, the CBH1 and CBH2 concentrations gradually rose as the reaction progressed, indicating desorption from the substrate. The final concentrations of CBH1 and CBH2 after the reaction were 80% and 90% of the original concentration respectively. Under the agitated condition in which CBH2 is deactivated, no increase in the concentration of CBH1 or CBH2 was observed after desorption. CBH2 is likely to be deactivated by shear stress from agitation. However, CBH1 is stable even under the agitated condition, indicating that, under agitated condition, CBH1 remains adsorbed to the substrate and the hydrolysis remains incomplete.

From this result, the exo-exo synergism was hypothesized as enhancing the hydrolysis by preventing the unproductive binding of CBH1 during the reaction. For further investigation, the adsorption and desorption characteristics of enzymes during the sequential addition of CBH1 and CBH2 fractions was observed. The hydrolysis of 10 wt% filter paper using CBH1 fraction yielded hydrolyzate with glucose concentration of 60 g/L at the end of the reaction, and no desorption of CBH1 from substrate was observed. The addition of CBH2 fraction at the later stage of this reaction reaccelerated the reaction, increasing the final glucose concentration to 90 g/L. Also, CBH1 desorption was observed after the addition of the CBH2 fraction.  The results obtained suggested that CBH1 desorbs from the substrate and completes the hydrolysis under the presence of active CBH2, but remains absorbed on the substrate in an inactive form when CBH2 is absent.

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