Comparative characterization of all cellulosomal cellulases from Clostridium thermocellum reveals high diversity in endoglucanase product formation essential for complex activity

Benedikt Leis,Katharina Dennemarck,Alarich Reiter,Wolfgang H Schwarz,Wolfgang Liebl,Sigrid Graubner,Robert Steinbauer,Matthias Moerch,Fabian Bergkemper,Matthias Mechelke,Claudia Held,Vladimir V Zverlov

doi:10.1186/s13068-017-0928-4

Abstract

BackgroundClostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels. It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA.ResultsAlthough all 24 single-cellulosomal cellulases were described previously, we present the first comparative catalogue of all these enzymes together with a comprehensive analysis under identical experimental conditions, including enzyme activity, binding characteristics, substrate specificity, and product analysis. In the course of our study, we encountered four types of distinct enzymatic hydrolysis modes denoted by substrate specificity and hydrolysis product formation: (i) exo-mode cellobiohydrolases (CBH), (ii) endo-mode cellulases with no specific hydrolysis pattern, endoglucanases (EG), (iii) processive endoglucanases with cellotetraose as intermediate product (pEG4), and (iv) processive endoglucanases with cellobiose as the main product (pEG2). These modes are shown on amorphous cellulose and on model cello-oligosaccharides (with degree of polymerization DP 3 to 6). Artificial mini-cellulosomes carrying combinations of cellulases showed their highest activity when all four endoglucanase-groups were incorporated into a single complex. Such a modeled nonavalent complex (n = 9 enzymes bound to the recombinant scaffolding protein CipA) reached half of the activity of the native cellulosome. Comparative analysis of the protein architecture and structure revealed characteristics that play a role in product formation and enzyme processivity.ConclusionsThe identification of a new endoglucanase type expands the list of known cellulase functions present in the cellulosome. Our study shows that the variety of processivities in the enzyme complex is a key enabler of its high cellulolytic efficiency. The observed synergistic effect may pave the way for a better understanding of the enzymatic interactions and the design of more active lignocellulose-degrading cellulase cocktails in the future.

Highlights

Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels
Exo-acting cellobiohydrolases hydrolyze the polysaccharide chain either from the reducing or non-reducing end, while endoglucanases cleave within the cellulose chain to generate new ends that are susceptible to subsequent hydrolysis by exoglucanase enzymes [2]
Characterizing the cellulosomal cellulases According to genome sequence analysis and to proteomics data of the extracellular cellulosomal complex of C. thermocellum [17, 18, 33, 54], in total 24 cellulaseencoding genes were selected for subsequent enzyme characterization (Table 1)

Summary

Introduction

Clostridium thermocellum is a paradigm for efficient cellulose degradation and a promising organism for the production of second generation biofuels It owes its high degradation rate on cellulosic substrates to the presence of supra-molecular cellulase complexes, cellulosomes, which comprise over 70 different single enzymes assembled on protein-backbone molecules of the scaffold protein CipA. Due to the complex structure of plant cell walls, biomassderived polysaccharides embody a rich tapestry of sugars and sugar compositions which are degraded by cellulases and other glycoside-depolymerizing enzymes. These enzymes can be described by three-dimensional structural analysis, sequence-based classification, substrate specificity, hydrolytic reaction mode, kinetic parameters, and product formation. Binding of the enzyme to the substrate requires the presence of specific carbohydratebinding modules (CBM) and sugar-binding residues on the enzyme surface and catalytic cleft

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Results

Discussion

Conclusion