THERMAL DENATURATION AND REFOLDING OF CARBOHYDRATE BINDING MODULES TO IMPROVE ENZYME RECYCLING IN A LIGNOCELLULOSIC BIOREFINERY

Abstract

Second generation bioethanol using lignocellulosic waste is a promising source of renewable energy. Cellulose, lignin, and other biopolymers make up lignocellulose. Saccharification of lignocellulose requires cellulases, which have a catalytic domain (CD) and a carbohydrate binding module (CBM) that binds the enzyme to substrates. Cellulase activity is known to decrease during saccharification when CBMs irreversibly bind lignin that the CD cannot hydrolyze for release and further reactions. While costly, cellulases must be supplemented after each cycle of saccharification. Here we demonstrate that following denaturation when heated to 5°C above the melting temperature (Tm), CBMs 11 and 44 (CAZy families) from Hungateiclostridium thermocellum (CtCBM11 and CtCBM44) can be released from a bound substrate. Once cooled to a temperature below the Tm, CtCBM11 and CtCBM44, Type B CBMs with a β-sandwich fold, spontaneously refold and regain binding function. Using temperature tunable CBMs could drastically reduce saccharification costs by improving enzyme recycling strategies.