Purification and characterization of two forms of the homologously expressed lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum

Two forms of C1/C4-oxidizing lytic polysaccharide monooxygenase (PvLPMO9A) from Penicillium verruculosum (Talaromyces verruculosus) homologously expressed in P. verruculosum B1-537 auxotrophic strain were isolated in a homogeneous state using two-stage chromatography. The PvLPMO9A-hm form represented a full-size enzyme encoded by the intact lpmo1 gene, while the PvLPMO9A-lm was a truncated enzyme variant consisting of a conserved catalytic core of AA9 family LPMOs and lacking a C-terminal extra peptide sequence that is present in PvLPMO9A-hm. The N-terminal histidine was partially methylated in both enzymes. Most of properties of PvLPMO9A-hm and PvLPMO9A-lm, such as specific activities determined using the 2,6-dimethoxyphenol/H₂O₂ assay, pH-optima of activity observed at pH 7.5, synergistic effects exhibited with purified cellobiohydrolase I (Cel7A) and/or endoglucanase II (Cel5A) from P. verruculosum in hydrolysis of Avicel and milled aspen wood, were also very similar, except for the higher PvLPMO9A-hm thermostability studied using differential scanning calorimetry (DSC). The DSC profile for the PvLPMO9A-hm holoenzyme demonstrated two overlapping peaks (with maxima at 56.3 and 59.6 °C) due to the presence of two unfolding protein domains, while the PvLPMO9A-lm DSC profile represented one peak with maximum at 48.1 °C. After removing the active site copper with EDTA, the PvLPMO9A-hm and PvLPMO9A-lm melting temperatures decreased by ~10–11 and ~1 °C, respectively. These data show that both active site copper and C-terminal domain present in the PvLPMO9A-hm protect the enzyme from thermal unfolding, while the stabilizing effect of metal is much less pronounced in the truncated PvLPMO9A-lm form.