Abstract
In recent years, bioeconomic strategies have gained much attention to maintain a sustainable environment and recycle waste and biomass. Lignocellulosic biomass isthe most abundant renewable source; it comprises cellulose (40-60%), hemicellulose (15-30%), and lignin. Enzymatic hydrolysis is a promising and sustainable technology that promotes bond cleavage and liberates oligomeric and monomeric carbohydrates from cellulose and hemicellulose. However, enzyme separation and reusability have been challenging as these methods are costly and sometimes lead to enzyme inactivation. One effective method is enzyme immobilization on a solid support matrix, i.e., magnetic nanoparticles. Immobilization of the catalysts provides easy separation and support to the enzyme and can be used repeatedly.
In this research study, four enzyme preparations, two commercials from Novozyme, Cellic CTec2 and NS81233, and one experimental EC200 (DSM), were investigated for their capacity to hydrolyze production fractions of steam-exploded spruce (Glommen Technology AS). Enzymatic hydrolysis was performed using 10 % (w/v) cellulose-enriched Cellin loading against different doses of enzyme Cellic CTec2, while hydrolysis of hemicellulose-enriched liquid wood molasses (WM) was performed using 10 % (v/v) loading against different doses of enzymes NS81233 and EC200. Samples were periodically withdrawn (0-24 hrs) and analyzed for reducing sugars (glucose or xylose equivalents) by DNS assay and on carbohydrate spectrum by RI-HPLC.
Cellin hydrolysis with Cellic CTec2 results showed that the overall saccharification yield increases with the enzyme dose; however, nonlinear, as 1% shows a 74% increase and 16% dose shows a 78% increase in GE concentration. NS81233 and EC200, the two enzymes, characterized for hemicellulose activity, were then used for the hydrolysis of WM. Even though NS81233 showed high potency against xylan as a hemicellulose substrate, the enzyme failed to exhibit any hydrolytic activity against spruce WM. The activity of the EC200 enzyme blend, on the other hand, demonstrated high mannanase catalytic activity against WM. A 1%(v/w) dose contributed to a 49% increase in mannose concentration, while an enzyme dose of 16%(v/w) doubled the mannose yield over the 1% dose.
EC200 enzyme was successfully immobilized on magnetic iron oxide nanoparticles (MNPs). The MNPs were prepared using ferric and ferrous chloride at a high temperature and then functionalized with 3-aminopropyl triethoxysilane (APTES). The enzyme was covalently linked with glutaraldehyde and analyzed for the extent of protein coupling with the Bradford Coomassie assay. The immobilized MNPs-EC200 were tested against 2% xylan substrate in ratios 1:1 and 1:1.5 (MNPs-EC200: xylan) and with 75 % (dry solids) WM in a ratio of 1:1 (MNPs-EC200: WM). The immobilized enzyme showed less catalytic activity than the free enzyme but yielded xylose (mg XE/ml) within a stable range after use in multiple saccharification processes with xylan (3) and with WM (20) as substrate. Low catalytic activity was due to the confinement of the enzyme to the nanoparticle’s surfaces. However, other factors may also affect the enzymatic activity, i.e., type of enzyme, nanoparticles, its shape and size, crosslinking agent, type of linkage formed, and substrate used.