(617c) Breaking the Barrier to Obtain High Yields On Enzymatic Hydrolysis Using Low Enzyme Loadings

A revolutionary method that enables efficient hydrolysis of cellulose under low enzyme loading was discovered through optimization of the mixing. Despite the simplicity of this method, it was never considered in previous studies. The application of this method allowed more than 10 fold reduction in the enzyme loading in hydrolysis of cellulosic biomass. The effect was confirmed when delignified biomass such as filter paper and pulp was used as a substrate. The previous studies revealed the following phenomenon; under a constant substrate loading, the final sugar concentration decreased when the enzyme loading was reduced. One would assume that if the saccharification ability of the enzyme is constant, lowering the enzyme loading will result in a longer reaction time, but not in a lower final glucose concentration. However, in the real reactions, the lower enzyme loadings resulted in lower final glucose concentrations. There hasn't been a clear explanation to this phenomenon yet, and it has been hindering us from reducing the enzyme loadings in the reaction. The method developed increased the final glucose concentration under lower enzyme loading, and gives an insight on an explanation to this phenomenon. The method optimized the mixing during the saccharification, and complex equipment or process isn't required. (The details will not be written in the abstract since the patent application is currently being filed). The studies showed that in case of the saccharification of 10% filter paper slurry using previous agitation method, the reaction with the enzyme loading of 79FPU/g-cellulose of Cellulase SS (Nagase ChemteX) achieved the final glucose concentration of 90g/L, while the enzyme loading of 7.9FPU/g-cellulose results in the final glucose concentration of 60g/L. Using the new method, the final glucose concentration of 90g/L was achieved with 7.9FPU/g-cellulose. The difference in the adsorption characteristic of the enzymes between the previous methods and the new method was also observed. Under the condition of 10w/v% filter paper, 79FPU/g-cellulose, enzyme concentrations in the hydrolyzate was measured. Using the previous method, the enzyme concentration decreased to 25% of the initial concentration at the beginning of the reaction. The enzyme concentration gradually recovered as the reaction progressed, and at the end of the reaction, the enzyme concentration in the hydrolyzate was about a half of the initial concentration. When the new method was used, the enzyme concentration decreased to 50% of the initial concentration at the beginning, and at the end of the reaction, the concentration was about 75% of the initial concentration. Discovery of this new method leaded to a foundation that the change in mixing procedures significantly affects the saccharification mechanism of cellulase. The further studies on the relationship between the mixing procedure and the saccharification mechanisms will result in not only improving the saccharification reaction process, but in the development of novel and more powerful enzymes. This new method may also challenge the previous assessments of the developed but underrated enzymes, which might show a significant improvement when this new method is applied. In the future studies, the application of the new method to a broader spectrum of biomass, such as pretreated corn stover, will be tested.