Although multicopper oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) can be particularly useful in biotechnological applications, e.g., as a specific catalyst at the biocathode of biofuel cells (BFCs), owing to its high stability against extremely high temperatures and across a wide range of pH values, this application potential remains limited due to the enzyme’s low catalytic activity. A directed evolution strategy was conducted to improve McoP catalytic activity, and the No. 571 mutant containing four amino acid substitutions was identified, with specific activity approximately 9-fold higher than that of the wild type enzyme. Among the substitutions, the single amino acid mutant F290I was essential in enhancing catalytic activity, with a specific activity approximately 12-fold higher than that of the wild type enzyme. F290I thermostability and pH stability were notably comparable with values obtained for the wild type. Crystal structure analysis suggested that the F290I mutant increased loop flexibility near the T1 Cu center, and affected electron transfer between the enzyme and substrate. Additionally, electric current density of the F290I mutant-immobilized electrode was 7-fold higher than that of the wild type-immobilized one.

These results indicated that F290I mutant was a superior catalyst with potential in practical biotechnological applications.

虽然来自超嗜热古细菌Pyrobaculum aerophilum 的多铜氧化酶(McoP) 在生物技术应用中特别有用，例如，作为生物燃料电池 (BFC) 生物阴极的特定催化剂，由于其对极高温度和广泛 pH 值的高稳定性，这种应用潜力仍然有限由于酶的催化活性低。采用定向进化策略提高McoP催化活性，鉴定出含有4个氨基酸置换的571号突变体，比活性比野生型酶高约9倍。在这些替换中，单氨基酸突变体 F290I 对增强催化活性至关重要，其比活性比野生型酶高约 12 倍。F290I 热稳定性和 pH 稳定性与野生型获得的值相当。晶体结构分析表明，F290I 突变体增加了 T1 Cu 中心附近的环柔性，并影响了酶和底物之间的电子转移。此外，F290I 突变体固定电极的电流密度比野生型固定电极的电流密度高 7 倍。

这些结果表明 F290I 突变体是一种在实际生物技术应用中具有潜力的优良催化剂。