Investigation of lignin effects on enzymatic hydrolysis of cellulosic substrates

Xiang Li, Clemson University


Lignocellulose is the most abundant raw material for biofuel production. Currently bioconversion of lignocellulose is a promising pathway to produce renewable biofuels and bio-based products, but still has several limitations, one of which is the inhibitory effect of lignin on enzymes. However, the underlying mechanisms are still not clear, and many inconsistent, and even contradictory, conclusions have been reached. The present study focused on the investigation of mechanisms of lignin-enzyme interactions and exploration of the definitive influences of different factors. Three types of isolated lignin were investigated, and both pure cellulose and lignocellulose were utilized as substrates for enzymatic hydrolysis. The results showed that different kinds of lignin affected enzymatic hydrolysis via different mechanisms. Organosolv loblolly pine (EOL LP) lignin presented high affinity to cellulase and inhibited enzymatic hydrolysis mainly by non-productive adsorption. Kraft pine lignin (KPL) interacted with cellulose and limited its accessibility to enzyme degradation, therefore impaired saccharification. During enzymatic hydrolysis process, decrease of rate and yield has been widely observed and generally a loss of enzyme activity and decrease in substrate reactivity have been observed. Lignin can affect enzymatic hydrolysis negatively by non-productive adsorption and/or steric repulsion, therefore the increase of lignin/cellulose mass ratio during hydrolysis may intensify the negative effect of lignin, resulting in the decrease of hydrolysis rate and yield. In addition, the reactivity of cellulose also changes during hydrolysis, e.g., increase of crystalline portion, thus reducing hydrolysis rate and yield. Using dilute acid pretreated switchgrass as an example, the effects of both lignin and cellulose on the enzymatic hydrolysis were investigated. Partially hydrolyzed switchgrass with different extents of conversion were prepared, and used as substrates for subsequent hydrolysis. These substrates at different stages of hydrolysis were evaluated in terms of composition, digestibility, enzyme adsorption capability and structural characteristics. A strong negative relationship was observed between lignin content and hydrolysis performance, which showed substrate with higher lignin content exhibited higher affinity and adsorption to enzyme. It was indicated that the increase of lignin content led to significant inhibition on hydrolysis process, and such inhibition was mainly due to nonproductive adsorption of enzyme by lignin. In addition, it was found that lignin adsorption of cellulase resulted in only partial enzyme activity loss.