Influences of the Carbohydrate-Binding Module on a Fungal Starch-Active Lytic Polysaccharide Monooxygenase.

Noncatalytic carbohydrate-binding modules (CBMs) play important roles in the function of lytic polysaccharide monooxygenases (LPMOs) but have not been well demonstrated for starch-active AA13 LPMO. In this study, four new CBMs were investigated systematically for their influence on Mt LPMO toward starch in terms of substrate binding, H2 O2 production activity, oxidative product yields, and the degradation effect with α-amylase and glucoamylase toward different starch substrates. Among the four Mt LPMO-CBM chimeras, Mt LPMO- Cn CBM harboring the CBM from Colletotrichum nymphaeae showed the highest substrate binding toward different types of starch compared to Mt LPMO without CBM. Mt LPMO- Pv CBM harboring the CBM from Pseudogymnoascus verrucosus and Mt LPMO- Cn CBM showed dramatically enhanced H2 O2 production activity of 4.6-fold and 3.6-fold, respectively, than Mt LPMO without CBM. More importantly, Mt LPMO-CBM generated more oxidative products from starch polysaccharides degradation than Mt LPMO alone, with 6.0-fold and 4.6-fold enhancement obtained from the oxidation of amylopectin and corn starch with Mt LPMO- Cn CBM, and a 5.2-fold improvement obtained with Mt LPMO- Ac CBM for amylose. Mt LPMO- Ac CBM significantly boosted the yields of reducing sugar with α-amylase upon degrading amylopectin and corn starch. These findings demonstrate that CBMs greatly influence the performance of starch-active AA13 LPMOs due to their enhanced binding and H2 O2 production activity.