Functional analysis of chimeric TrCel6A enzymes with different carbohydrate binding modules

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.