Exploring methods for functional studies of CBM33-type lytic polysaccharide monooxygenases

Abstract

Conversion of biomass to sustainable sources of soluble sugars for fermentation has been subject of intensive research in the field of biorefinery the last decade. The recalcitrant nature of many polysaccharides create a technical barrier to the cost-effective transformation of biomass to fermentable sugars, but a novel discovery have given more insight into efficient conversion of recalcitrant polysaccharides. Bacterial proteins categorized as Lytic Polysaccharide Monooxygenases (LPMO) member of family 10 auxiliary activities (AA) (former family 33 carbohydrate-binding modules) were recently shown to boost degradation of crystalline chitin using a mechanism involving an oxidative and a hydrolytic step. A better understanding of chitin degradation can be used for optimizing conditions for degradation of other polysaccharides in a crystalline context, such as cellulose. The aim of this study was to gain more insight into the binding properties and the catalytic mechanism of chitin-binding protein 21 (CBP21), a surface-active AA10-type LPMO, produced by the well known chitinolytic soil bacterium Serratia marcescens. The functional role of seven conserved residues located close to the active site (Ser-58, Thr-111, Gly-112, Trp-178, Ile-180, Thr-183 and Phe-187) were probed by site-directed mutagenesis, binding assays and product analysis. All mutations lowered the affinity for CBP21 to β-chitin and their ability to degrate the substrate, but CBP21 variant I180R showed a substantially lower activity than the wild-type and the mutants. A novel assay to measure enzyme activity based on detection of hydrogen peroxide from a futile side reaction of LPMOs was tested on CBP21, showing that the copper present in the enzyme contributes to formation of hydrogen peroxide by autooxidation. In addition, various AA10s known to interact with the human gut were tested for substrate specificity by a glycan array screen. GbpA from Vibrio cholerae was the only enzyme that showed binding, an enzyme which was also proven to have LMPO activity.