The activity and stability analyses of chitin-activity lytic polysaccharide monooxygenase

Abstract:

[Objective] Lytic polysaccharide monooxygenase (LPMO) is a recently discovered copper ion-dependent oxidase, which can break glycosidic bonds by oxidation, thus significantly improving the efficiency of polysaccharides degradation. However, it is easy to be inactivated and difficult to evaluate the activity of LPMO due to the properties of its substrates and the diversity of its released products.[Methods] In this study, we established a spectrophotometric activity assay to detect the chitin-active LPMO (BtLPMO10A) using 2,6-dimethoxyphenol (2,6-DMP) and H₂O₂ as substrates, and to evaluate the stability of LPMO during chitin degradation.[Results] The results suggested that high concentration of enzyme, 2,6-DMP or H₂O₂ would deviate the reaction from linear range. The K_m of BtLPMO10A toward 2,6-DMP and H₂O₂ was determined to be 0.53 mmol/L and 5.31 mmol/L respectively. It suggested BtLPMO10A possessed a higher affinity toward 2,6-DMP and H₂O₂ than NcLPMO9C. BtLPMO10A was easy to be inactivated in the presence of reducing agent ascorbic acid. The substrate chitin could stabilize the enzyme, but the activity still decreased during the degradation of chitin.[Conclusion] This work evaluated the factors that impacted on the assay for detecting the activity of BtLPMO10A using 2,6-DMP as substrate, and estimated the stability of BtLPMO10A during chitin degradation. It will provide important information for the investigation of chitin-active LPMOs.