Discovery and functional characterization of new starch-active lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) play a unique role in biomass saccharification as they catalyze oxidative cleavage of recalcitrant polysaccharides. Here, four new starch-active AA13 LPMOs were identified via phylogenetic analysis and functionally expressed in Pichia pastoris, followed by the investigation of oxidative activity towards different starch substrates. The four purified AA13 LPMOs were capable of oxidizing amylose, amylopectin and corn starch, generating soluble products. Among the four, FvAA13, which exhibited the best oxidative activity, was further studied. Its yield of oxidized products was 1.9-fold, 1.4-fold, and 1.3-fold higher than the previously reported NcAA13 when applied to starch substrates amylose, amylopectin, and corn starch, respectively. Furthermore, the oxidized product yield of FvAA13 was enhanced by 2-fold when supplemented with 100 μM H₂O₂. FvAA13 also enhanced the amylose degradation catalyzed by α-amylase and glucoamylase at 50 °C with an increment of the product by 1.4-fold and 1.9-fold, respectively. When combined with glucoamylase at a mass concentration ratio of 0.5:1 to 2:1, the reducing sugars released from the enzymatic degradation of amylopectin were improved by 4.5 to 9.7-fold compared to using glucoamylase alone. These findings underscored the substantial promoting effect of the supplementary FvAA13 in amylolytic cocktails for starch saccharification.