Functional analysis of chimeric TrCel6A enzymes with different carbohydrate binding modules

Stefan Jarl Christensen, Silke Flindt Badino, Ana Mafalda Cavaleiro, Kim Borch, Peter Westh*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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.
Original languageEnglish
Article numbergzaa003
JournalProtein Engineering Design and Selection (Print)
Volume32
Issue number9
Pages (from-to)401-409
Number of pages9
ISSN1741-0126
DOIs
Publication statusPublished - 2020

Keywords

  • Cellulases
  • CBM
  • GH6
  • Cellulose
  • Chimeras
  • Cel6A

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