Enhanced features of Dictyoglomus turgidum Cellulase A engineered with carbohydrate binding module 11 from Clostridium thermocellum

Chiara Cattaneo,Sara Icardi,Patrizia Cesaro,Maria Cavaletto,Stefano Spertino

doi:10.1038/s41598-018-22769-w

Chiara Cattaneo, Sara Icardi + Show 3 more

Open Access

https://doi.org/10.1038/s41598-018-22769-w

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Abstract

Lignocellulosic biomass (LCB) is a low-cost and abundant source of fermentable sugars. Enzymatic hydrolysis is one of the main ways to obtain sugars from biomass, but most of the polysaccharide-degrading enzymes are poorly efficient on LCB and cellulases with higher performances are required. In this study, we designed a chimeric protein by adding the carbohydrate binding module (CBM) of the cellulosomal enzyme CtLic26A-Cel5E (endoglucanase H or CelH) from Clostridium (Ruminiclostridium) thermocellum to the C-terminus of Dtur CelA, an interesting hyperthermostable endoglucanase from Dictyoglomus turgidum. The activity and binding rate of both native and chimeric enzyme were evaluated on soluble and insoluble polysaccharides. The addition of a CBM resulted in a cellulase with enhanced stability at extreme pHs, higher affinity and activity on insoluble cellulose.

Highlights

Lignocellulosic biomass (LCB) represents an invaluable source of “green energy” providing fermentable sugars for different purposes, among which the production of bioethanol
We designed a chimeric protein by adding the CBM11 and linker peptide from CtCelH to the C-terminus of Dtur CelA
carbohydrate binding module (CBM) 11 from Clostridium thermocellum has been chosen, since it is naturally linked to a catalytic domain (Cel5E) classified as GH5 in the cellulosomal enzyme CelH

Summary

Introduction

Lignocellulosic biomass (LCB) represents an invaluable source of “green energy” providing fermentable sugars for different purposes, among which the production of bioethanol. Most cellulases contain non-catalytic carbohydrate-binding modules (CBMs) connected to the catalytic domain (CD) through a linker peptide that is sometimes highly flexible[7]. It is known that CBMs recognize different polysaccharides in a specific way, the CBMs present in cellulases bind cellulose, enhancing enzyme activity on the insoluble polysaccharide[9,10]. C. thermocellum Lic26A-Cel5E ( known as endoglucanase H or CelH; UniprotKB: P16218) is a cellulosomal enzyme containing two catalytic modules (Lic26A and Cel5E), a CBM11 and two C-terminal type I dockerin domains. D. turgidum CelA has no electronically detectable CBM, as previously reported[6], we designed a chimeric protein by adding the CtCBM11 to the C-terminus of Dtur CelA catalytic domain, connecting them by a linker peptide from CtLic26A-Cel5E. The aim of this study was to evaluate the effects of protein engineering on the activity and binding rate of Dtur CelA on both soluble and insoluble polysaccharides

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