Quantitative analysis of adsorption and desorption behavior of individual cellulase components during the hydrolysis of lignocellulosic biomass with the addition of lysozyme.

The effect of non-catalytic protein addition on the adsorption/desorption behavior of individual cellulase components on/from substrates during the hydrolysis of microcrystalline cellulose and steam exploded sugarcane bagasse (SEB) were investigated. The addition of non-catalytic protein enhanced the enzymatic hydrolysis of SEB, but did not enhance the hydrolysis of microcrystalline cellulose. During the hydrolysis of SEB, adsorption of beta-glucosidase (BGL) was prevented in the presence of non-catalytic protein. Cellobiohydrolase I (CBH I) and endoglucanase I (EG I) desorbed from the substrate after temporary adsorption in the presence of non-catalytic protein during SEB hydrolysis. This suggested that reduction of the non-specific adsorption of cellulase components, CBH I, EG I, and BGL, on lignin in SEB led to the improving of enzymatic hydrolysis.

Effects of Tween 80 on cellulase stability under agitated conditions.

The mechanism of the increase in the hydrolysis rate and yield by the addition of Tween 80 to the hydrolysis reaction of filter paper was investigated under static and agitated conditions. The increase in the hydrolysis rate by addition of Tween 80 was observed under the agitated condition only. The effects of Tween 80 on the changes in the protein concentration of individual cellulase components were investigated in the absence of substrates. Agitation of the enzyme solution resulted in the drastic decrease of SDS-PAGE bands intensity of CBH2 (cellobiohydrolase 2). The addition of Tween 80 prevented this. Thus, the Tween 80 functions to stabilize instable cellulase components under the agitated condition. Moreover, addition of Tween 80 completely suppressed the decrease of CBH2 intensity by agitation at 30°C. Results suggest that Tween 80 stabilizes instable cellulase components not only during hydrolysis, but during enzyme production also.

Characteristics of enzyme hydrolysis of cellulose under static condition.

The effect of enzyme loading under static and agitated conditions was investigated. Enzymatic hydrolysis of 10 w/v% de-lignified cellulose slurry such as filter paper, avicel and pulp was conducted under agitated (120 rpm) and static condition, and the enzyme loading ranging from 1.2 to 120 mg-protein/g-dry substrate. Under the agitated condition, the final sugar concentration decreased with the decreasing enzyme loading. Under the static condition, the final sugar concentration was maintained even if the enzyme loading was decreased. The above phenomenon was caused by a rapid precipitation of cellobiohydrolase 2 (CBH2) under the agitated condition, which was not observed under the static condition. The hydrolysis experiments using enzymes containing different ratios of cellobiohydrolase 1 (CBH1) and CBH2 under the static condition suggested that preservation of CBH2 and its synergism with CBH1 is essential for static condition's characteristics, and for efficient hydrolysis of cellulose.