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, H 2 O 2 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 H 2 O 2 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 H 2 O 2 production activity.