Adsorption characteristics of enzymes on lignocellulosic material by liquid chromatography

Our previous work demonstrated that severe pretreatment not only opens up the structure for enzymatic hydrolysis, but also increases lignin surface area exposed to cellulases. Non-productive binding of cellulases onto lignin decrease thier activity. Therefore, higher enzyme loading is required to compensate for loss of enzyme due to adsorption on lignin. Previous reports have shown that BSA is effective in adsorbing onto lignin and blocking exposed lignin surface agianst adsorption of cellulase enzymes, thus increasing the effectiveness of enzymatic hydrolysis. Further studies on competitive adsorption of BSA and enzyme are now being carried out to better understand the lignin blocking effects. The traditional method of determining adsorption parameters for enzyme-lignin interactions through batch-adsorption studies is time consuming and labor intensive. Therefore an inverse liquid chromatography method was developed instead, in order to determine the protein adsorption characteristics of lignin and lignocellulosic solids packed in a chromatography column. In this study, sugarcane bagasse was the stationary phase. Preliminary results observed by injecting 500 µL of BSA (20 mg/mL) showed that BSA is retained in the column with a retention time of 17.6 min at both 20 and 50°C, although sharper peaks were observed at 50°C, consistent with the Arrhenius definition of the temperature dependence of an adsorption constant. These results confirmed the expected adsorption behavior of BSA, but more importantly, illustrated the utility of inverse liquid chromatography to better understand the adsorption of cellulases and other proteins to lignin. Inverse chromatography is being developed further as a rapid screening process for potential lignin blocking proteins.