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.

中文翻译：

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来自青霉菌的两种形式的同源表达的裂解性多糖单加氧酶（PvLPMO9A）的纯化和表征。

使用两步色谱法，以均相状态分离了在绿假单胞菌B1-537营养缺陷型菌株中同源表达的两种来自青霉（Talaromyces verruculosus）的C1 / C4氧化型溶解性多糖单加氧酶（PvLPMO9A）。PvLPMO9A-hm形式代表了完整的lpmo1基因编码的完整酶，而PvLPMO9A-lm是一种截短的酶变体，由AA9家族LPMO的保守催化核心组成，并且缺少C端额外的肽序列在PvLPMO9A-hm中。N末端组氨酸在两种酶中均被部分甲基化。PvLPMO9A-hm和PvLPMO9A-lm的大多数特性，例如使用2,6-二甲氧基苯酚/ H2O2测定法测定的比活，在pH 7.5时观察到的最佳pH活性，与绿疣假单胞菌的纯化纤维二糖水解酶I（Cel7A）和/或内切葡聚糖酶II（Cel5A）在Avicel和磨碎的白杨木水解中表现出的协同作用也非常相似，不同之处在于使用差示扫描量热法研究了更高的PvLPMO9A-hm热稳定性（ DSC）。由于存在两个未折叠的蛋白结构域，PvLPMO9A-hm全酶的DSC谱显示两个重叠的峰（最大值在56.3和59.6°C），而PvLPMO9A-lm DSC谱代表一个峰，在48.1°C时有最大值。用EDTA除去活性部位的铜后，PvLPMO9A-hm和PvLPMO9A-lm的熔化温度分别降低了约10-11和约1°C。这些数据表明，PvLPMO9A-hm中存在的活性位点铜和C末端结构域均可保护酶免于热解折叠，