Heterologous expression and characterization of Aspergillus oryzae lytic polysaccharide monooxygenases

Abstract:

[Objective] Lytic polysaccharide monooxygenases (LPMOs) are new lignocellulose-degrading enzymes that break glycosidic bonds of polysaccharides via oxidation. This study aims to explore and characterize novel LPMOs. [Methods] LPMO genes were cloned from Aspergillus oryzae and expressed in Pichia pastoris. [Results] Sequence analysis indicated that both AoLPMO2 and AoLPMO5 belong to auxiliary activity 9 family. AoLPMO5 was transformed into P. pastoris GS115 to obtain a 2-copy insertion yeast GS/AO5-4. After induction by 1% methanol for 4 d, protein yield in culture supernatant reached 0.19±0.01 g/L. The molecular mass of recombinant protein was approximate 34 kDa, which was higher that the theorical calculation, indicating probably post-translation modification proceeded in yeast host. The optimal temperature and pH of AoLPMO5 to catalyze locust bean gum were 60℃ and 5.0. Its K_m and V_max were 8.72±1.99 mg/mL and 109.4±12.8 μmol/(s·mg), respectively. Addition of 0.1 mmol/L Cu²⁺, 0.5, 2.0 and 2.5 mmol/L H₂O₂ enhanced its catalytic activity by (7.10±1.32)% (P<0.05), (21.11±6.17)% (P<0.01), (20.22±1.13)% (P<0.01) and (18.40±2.86)% (P<0.01), respectively, while gallic acid or vitamin C did not show promotion. The AoLPMO5 was found to bind with locust bean gum substrate at the early stage of reaction, resulting in temporary inhibition of mannanase (BsMAN3) activity. However, these two enzymes showed synergic effects on substrate deconstruction at the later period. [Conclusion] AoLPMO5 is a novel biomass-degrading enzyme. Insights into its enzymatic properties and substrate-degrading patterns will contribute to natural lignocellulosic biomass conversion and biorefinery, especially second-generation bioethanol and functional oligosaccharides production.