7-13: Inverting enzymes produce less ethyl xylopyranoside than retaining enzymes during simultaneous saccharification and fermentation

Simultaneous saccharification and fermentation is a one tank process, in which ethanol is produced from biomass hydrolysis products while being formed. During SSF studies, we have identified a side product - ethyl xylopyranoside. We found that Trichoderma reesei family 3 and 10 b-xylosidases, like other T. reesei enzymes, are responsible for the EXP formation. On the other hand, we were able to demonstrate that adding a family 43 b-xylosidase to the mixture leads to a decrease in EXP formation and an increase of ethanol yield.

Glycosyl hydrolase enzymes generally act in one of two mechanisms – inversion or retention. The former is a one-step mechanism in which two carboxylate residues act as acid and base . The latter is a two-step mechanism in which two enzyme-borne carboxylates act as a nucleophile and an acid/base.

When ethanol competes with water in the reaction, ethyl-xylosides can be formed by two reactions – transglycosylation and reverse hydrolysis. Inverting enzymes, will produce ethyl-alpha-xyloside by transglycosylation and ethyl-beta-xyloside by reverse hydrolysis, while retaining enzymes, produce ethyl-b-xyloside by both transglycosylation and reverse hydrolysis.

Enzymes belonging to GH3 and GH10 are retaining, and were found to produce EXP and xylose rapidly from xylobiose by transglycosylation. Enzymes belonging to GH43, on the other hand, are inverting enzyme and produced EXP much slower, by reverse hydrolysis. In addition, it was observed that only the β-anomer is produced in this process. Therefore, the retaining GH3 and GH10 xylanases are mainly responsible for the EXP-formation and EXP-formation can be reduced by inverting enzymes.