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Functional analysis of chimeric TrCel6A enzymes with different carbohydrate binding modules.

Authors
  • Christensen, Stefan Jarl1
  • Badino, Silke Flindt1
  • Cavaleiro, Ana Mafalda1, 2
  • Borch, Kim2
  • Westh, Peter3
  • 1 Research Unit for Functional Biomaterials, Department of Science and Environment, Roskilde University, building 28B, DK-4000, Roskilde, Denmark. , (Denmark)
  • 2 Novozymes A/S, Department of Enzyme Discovery, Rævehøjvej 32A, DK-2800 Kgs. Lyngby, Denmark. , (Denmark)
  • 3 Department of Biotechnology and Biomedicine, Technical University of Denmark, building 224, DK-2800, Kgs. Lyngby, Denmark. , (Denmark)
Type
Published Article
Journal
Protein Engineering Design and Selection
Publisher
Oxford University Press
Publication Date
Dec 31, 2019
Volume
32
Issue
9
Pages
401–409
Identifiers
DOI: 10.1093/protein/gzaa003
PMID: 32100026
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

The glycoside hydrolase (GH) family 6 is an important group of enzymes that constitute an essential part of industrial enzyme cocktails used to convert lignocellulose into fermentable sugars. In nature, enzymes from this family often have a carbohydrate binding module (CBM) from the CBM family 1. These modules are known to promote adsorption to the cellulose surface and influence enzymatic activity. Here, we have investigated the functional diversity of CBMs found within the GH6 family. This was done by constructing five chimeric enzymes based on the model enzyme, TrCel6A, from the soft-rot fungus Trichoderma reesei. The natural CBM of this enzyme was exchanged with CBMs from other GH6 enzymes originating from different cellulose degrading fungi. The chimeric enzymes were expressed in the same host and investigated in adsorption and quasi-steady-state kinetic experiments. Our results quantified functional differences of these phylogenetically distant binding modules. Thus, the partitioning coefficient for substrate binding varied 4-fold, while the maximal turnover (kcat) showed a 2-fold difference. The wild-type enzyme showed the highest cellulose affinity on all tested substrates and the highest catalytic turnover. The CBM from Serendipita indica strongly promoted the enzyme's ability to form productive complexes with sites on the substrate surface but showed lower turnover of the complex. We conclude that the CBM plays an important role for the functional differences between GH6 wild-type enzymes. © The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]

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