Enzyme-substrate interactions and their influence on enzyme recycling strategies as a way of reusing cellulases

Abstract

Relatively high enzyme loadings are required for the bioconversion of lignocellulosic biomass, impeding the economical production of cellulosic sugars. The relative stability and robustness of these enzymes make enzyme recycling an attractive cost-reduction strategy. However, the efficiency of enzyme recycling has been limited by the complexity of enzyme-substrate interactions, which are influenced by enzyme, substrate, and physical factors. A lack of techniques to probe specific enzyme adsorption further limits our understanding of these interactions. Therefore, overcoming these challenges to better understand enzyme-substrate interactions is crucial if we are to improve the effectiveness of enzyme recycling strategies. Initial work compared various ways to assess enzyme adsorption during hydrolysis of steam pretreated corn stover (SPCS) using a complete commercial cellulase mixture. While the distribution of six individual enzymes could be followed, the initial approach used was laborious, highlighting the limitations of techniques used to quantify individual enzyme adsorption profiles. A quicker, more sensitive double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was subsequently developed, to follow Cel7A, Cel6A, and Cel7B adsorption during hydrolysis, and shown to agree with earlier results. As enzyme, substrate, and physical factors were known to affect enzyme recycling performance, their influence on individual enzyme adsorption was evaluated. Although the lignin present in the SPCS did not appear to influence enzyme adsorption (although Cel6A adsorbed more readily to the lignin-containing SPCS), cellulose allomorphs and crystallinity did appear to influence enzyme adsorption. The addition of Auxiliary Activity (AA) family 9, an oxidative enzyme, increased desorption of Cel7A, likely by increasing the substrate’s negative charge. The AA9 itself remained primarily in the supernatant, which highlighted the importance of recovering enzymes from both the liquid and solid phases of the reaction. The influence of glucose and ethanol on enzyme adsorption was evaluated, and a reduction in enzyme adsorption was observed at high glucose but not ethanol concentrations. When the addition of fresh substrate was assessed as one way to recover enzymes, by combining enzyme recycling at low glucose concentrations with enzyme supplementation, good overall cellulose hydrolysis (~70%) over 5 rounds of enzyme recycle could be achieved with a 50% reduction in enzyme loading.