Plant carbohydrate binding module enhances activity of hybrid microbial cellulase enzyme.

Caitlin S Byrt,Ricky Cahyanegara,Christopher P L Grof

doi:10.3389/fpls.2012.00254

Abstract

A synthetic, highly active cellulase enzyme suitable for in planta production may be a valuable tool for biotechnological approaches to develop transgenic biofuel crops with improved digestibility. Here, we demonstrate that the addition of a plant derived carbohydrate binding module (CBM) to a synthetic glycosyl hydrolase improved the activity of the hydrolase in releasing sugar from plant biomass. A CEL-HYB1-CBM enzyme was generated by fusing a hybrid microbial cellulase, CEL-HYB1, with the CBM of the tomato (Solanum lycopersicum) SlCel9C1 cellulase. CEL-HYB1 and CEL-HYB1-CBM enzymes were produced in vitro using Pichia pastoris and the activity of these enzymes was tested using carboxymethylcellulose, MUC, and native crystalline cellulose assays. The presence of the CBM substantially improved the endoglucanase activity of CEL-HYB1, especially against the native crystalline cellulose encountered in Sorghum bicolor plant cell walls. These results indicate that addition of an endogenous plant derived CBM to cellulase enzymes may enhance hydrolytic activity.

Highlights

Increasing the efficiency of enzymatic hydrolysis of cellulose is a key challenge in the process of reducing the cost of producing transport fuels from lignocellulosic biomass (Cheng et al, 2011)
ENGINEERING OF A GLYCOSYL HYDROLASE ENZYME In order to develop a highly active glycosyl hydrolase suitable for production in planta the nucleotide sequence of a highly active endo-β-1,4-glucanase of microbial origin, previously codon optimized for plants (Xue et al, 2003) was fused directly to the nucleotide sequence encoding a carbohydrate binding module (CBM) of plant origin
Www.frontiersin.org rich linker region between the catalytic domain (CD) and the CBMs (Batista et al, 2011); no linker region was included between CEL-HYB1 and the CBM in this study

Summary

Introduction

Increasing the efficiency of enzymatic hydrolysis of cellulose is a key challenge in the process of reducing the cost of producing transport fuels from lignocellulosic biomass (Cheng et al, 2011). One factor influencing the efficiency of hydrolysis is the interaction of soluble cellulase enzymes with the insoluble cellulose (Cheng et al, 2011). Cellulase enzymes belong to different families of glycoside hydrolases (GHs). Many cellulases contain a catalytic domain (CD) and a cellulose binding domain or module (Linder and Teeri, 1997); these are connected by a Pro/Ser/Thr rich linker (Batista et al, 2011). Other domains may be present and these were recently described for bacterial cellulases in a comprehensive genome analysis (Medie et al, 2012)

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Results

Conclusion