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 MtLPMO toward starch in terms of substrate binding, H2O2 production activity, oxidative product yields, and the degradation effect with α-amylase and glucoamylase toward different starch substrates. Among the four MtLPMO-CBM chimeras, MtLPMO-CnCBM harboring the CBM fromColletotrichum nymphaeae showed the highest substrate binding toward different types of starch compared to MtLPMO without CBM. MtLPMO-PvCBM harboring the CBM from Pseudogymnoascus verrucosus and MtLPMO-CnCBM showed dramatically enhanced H2O2 production activity of 4.6-fold and 3.6-fold, respectively, than MtLPMO without CBM. More importantly, MtLPMO-CBM generated more oxidative products from starch polysaccharides degradation than MtLPMO alone, with 6.0-fold and 4.6-fold enhancement obtained from the oxidation of amylopectin and corn starch with MtLPMO-CnCBM, and a 5.2-fold improvement obtained with MtLPMO-AcCBM for amylose. MtLPMO-AcCBM 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 H2O2 production activity.
Keywords: carbohydrate-binding module (CBM); lytic polysaccharide monooxygenase (LPMO); oxidized products; starch binding; starch degradation.