Auxiliary activity 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) are copper-dependent oxidoreductases that use O2 or H2O2 to perform oxidative cleavage of cellulose in the presence of an electron donor. Combined with cellulases, they can assist in a more efficient cleavage of cellulose. AA9 LPMOs have therefore attracted considerable attention in recent years for use in biotechnological applications. Here, a native AA9 LPMO (nTaAA9A) from the thermophilic fungus Thermoascus aurantiacus was purified and characterized. The enzyme was shown to be active and able to cleave cellulose and xylan to produce C1- and C4-oxidized products. It was also found to retain about 84.3, 63.7, and 35.3% of its activity after incubation for 30 min at 60, 70, and 80 °C, respectively, using quantitative activity determination. The structure was determined to 1.36 Å resolution and compared with that of the recombinant enzyme expressed in Aspergillus oryzae. Structural differences in the glycosylated Asn138 and in solvent-exposed loops were identified.

Background Lytic polysaccharide monooxygenases (LPMOs) belonging to the auxiliary activity 9 family (AA9) are widely found in aerobic fungi. These enzymes are O2-dependent copper oxidoreductases that catalyze the oxidative cleavage of cellulose. However, studies that have investigated AA9 LPMOs of aerobic fungi in the herbivore gut are scare. To date, whether oxidative cleavage of cellulose occurs in the herbivore gut is unknown. Results We report for the first time experimental evidence that AA9 LPMOs from aerobic thermophilic fungi catalyze the oxidative cleavage of cellulose present in the horse gut to C1-oxidized cellulose and C1- and C4-oxidized cello-oligosaccharides. We isolated and identified three thermophilic fungi and measured their growth and AA9 LPMO expression at 37 °C in vitro. We also assessed the expression and the presence of AA9 LPMOs from thermophilic fungi in situ. Finally, we used two recombinant AA9 LPMOs and a native AA9 LPMO from thermophilic fungi to cleave cellulose to yield C1-oxidized products at 37 °C in vitro. Conclusions The oxidative cleavage of cellulose occurs in the horse gut. This finding will broaden the known the biological functions of the ubiquitous LPMOs and aid in determining biological significance of aerobic thermophilic fungi.

IntroductionMost current methods for analysing the activity of LPMO are based on the quantification of H2O2, a side product of LPMO; however, these methods cannot assay the LPMO activity of thermophilic fungi because of the low thermostability of H2O2. Therefore, we present a high-performance liquid chromatography–refractive index detector (HPLC-RID) method to assay the LPMO activity of the thermophilic fungus Thermoascus aurantiacus.ResultsAccording to the established method, the specific activities of nTaAA9A C1 and C4 oxidation were successfully analysed and were 0.646 and 0.574 U/mg, respectively. By using these methods, we analyzed the C1 and C4 oxidation activities of the recombinant TaAA9A (rTaAA9A) and mutated rTaAA9A (Y24A, F43A, and Y212A) expressed in Pichia pastoris. The specific activities of rTaAA9A C1 and C4 oxidation were 0.155 and 0.153 U/mg, respectively. The specific activities of Y24A, F43A, and Y212A C1 and C4 oxidation were 0.128 and 0.125 U/mg, 0.194 and 0.192 U/mg, and 0.097 and 0.146 U/mg, respectively.DiscussionIn conclusion, the method can assay the LPMO activity of thermophilic fungi and directly target C1 and C4 oxidation, which provides an effective activity assay method for LPMOs of thermophilic fungi.