(626i) Identification of the Cellulase Component That Contributes to the Efficient Saccharification of Cellulose At Low Enzyme Loadings

At the 2010 AIChE annual meeting, JGC Corporation reported on a method that enables efficient saccharification of cellulosic biomass at low enzyme loadings. In the report, the solution to the following phenomenon, which had been frequently observed in past studies, was presented: “Under a constant substrate loading, the saccharification of cellulose will reach its maximum yield when sufficient enzyme loading is used. However, when the enzyme loading is reduced, the yield of the reaction plateaus before it reaches the maximum yield.” This phenomenon will be termed as the “Plateau Phenomenon” in this paper. JGC Corporation demonstrated that the Plateau Phenomenon can be avoided by putting the saccharification reaction under the static condition. When the saccharification of 100ml 10% filter paper slurry was conducted under agitated condition, the resulting glucose concentration was 90g/L at the enzyme loading of 600mg Cellulase SS (Nagase Chemtex). At the enzyme loading of 60mg, the glucose concentration plateaued at 60g/L. However, by putting the reaction under the static condition, the final glucose concentration of 90g/L was achieved even at the enzyme loading of 60mg. This avoidance of the Plateau Phenomenon at lower enzyme loading and under the static condition was defined as the “Static Effect,” and the inactivation of the enzyme by agitation was the suspected cause.

 In this study, JGC Corporation identified a particular component of the cellulase that causes the Static Effect. This component is drastically inactivated by agitation. When the enzyme solution without any substrate was agitated at 115rpm and a temperature of 50°C, 80% of this component was lost from the solution within the first day, indicating its inactivation. The saccharification of cellulosic biomass was conducted using this “component-inactivated” enzyme solution. As a result, the enzyme solution in which this component is inactivated didn’t show a significant difference between the static and agitated condition. In other words, the enzyme solution without this component lost its ability to exhibit the Static Effect. Thus, this component was deduced to have a key role in cellulose saccharification, and it was identified by LC/MS analysis. This component will be defined as the “Key Enzyme” from here on. (The identity of this enzyme will not be given in the abstract since the patent application is currently being filed)

To confirm the effect of the Key Enzyme, the enzyme solution was fractionated using a HiLoad Q Sepharose HP 26/10 column. Some of the fractions obtained consisted mainly of the Key Enzyme. Using these fractions, enzyme solutions with varied ratios of the Key Enzyme to CBH1 was prepared, and saccharification experiments under a constant protein loading and the static condition were conducted. The results showed that, as the ratio of the Key Enzyme against CBH1 increased, the rate and the yield of the reaction increased. Moreover, when only the CBH1 was used for saccharification, the Plateau Phenomenon was observed, but by addition of the Key Enzyme, the Static Effect was observed. When a sufficient amount of the Key Enzyme was added, the Plateau Phenomenon was avoided. These results confirmed that the synergism between the Key Enzyme and CBH1 can overcome the Plateau Phenomenon.

A number of past studies reported the synergism between the individual cellulase components, especially those with CBH1. However, the synergism between CBH1 and the Key Enzyme reported in this study can only be confirmed under the static condition. Thus, in the past studies which were conducted under agitated conditions, the function of the key enzyme and its degree of synergism with CBH1 may have been underrated since the enzyme was easily inactivated by the shear force exerted by agitation. 

Further investigation of the role and properties of the Key Enzyme is necessary for effective saccharification of cellulose, and commercialization of the enzymatic saccharification process.