Efficient biotransformation of lignin requires the activity of different oxidative enzymes. In this work, 19 bacterial multi-copper oxidases were screened for oxidase activity against 19 soluble substrates and revealed the highest activity in the laccase CotA_Bsu (BSU0630) from Bacillus subtilis. Structure-based site-directed mutagenesis of CotA_Bsu identified four conserved residues (His419, Cys492, His497, and Met502) as critical for activity against 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS). Greatly reduced oxidase activity was found in the CotA_Bsu mutant proteins E213A, N214A, C229A, N264A, E298A, T415A, R416A, Q468A, and T480A. We also designed a lignin-agarose plate screen for detecting oxidase activity of purified proteins against polymeric lignin, which confirmed the results obtained with ABTS and identified three mutant variants with increased activity toward kraft lignin (E213A, T415A, and T260A). X-ray photoelectron spectroscopy analysis of low sulfonate kraft lignin after incubation with CotA_Bsu revealed a reduction in the content of C-C/C = C bonds and increase in C-O/C = O bonds. Product analyses using mass spectrometry, liquid chromatography, and bright-field microscopy revealed an increased polymerization state of reaction products suggesting that formation of radical intermediates was followed by radical coupling. Our results provide further insights into the mechanisms of lignin oxidation by laccases.

木质素的有效生物转化需要不同氧化酶的活性。在这项工作中，针对 19 种可溶性底物的氧化酶活性筛选了 19 种细菌多铜氧化酶，并揭示了来自枯草芽孢杆菌的漆酶 CotA _Bsu (BSU0630)的最高活性。CotA _Bsu的基于结构的定点诱变鉴定了四个保守的残基（His419、Cys492、His497 和 Met502），它们对于 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) 的活性至关重要。在 CotA _{Bsu 中}发现氧化酶活性大大降低突变蛋白 E213A、N214A、C229A、N264A、E298A、T415A、R416A、Q468A 和 T480A。我们还设计了一种木质素-琼脂糖板筛选，用于检测纯化蛋白质对聚合木质素的氧化酶活性，这证实了用 ABTS 获得的结果，并鉴定了三种对硫酸盐木质素活性增加的突变体（E213A、T415A 和 T260A）。与 CotA _Bsu孵育后低磺酸盐硫酸盐木质素的 X 射线光电子能谱分析揭示了 CC/C = C 键含量的减少和 CO/C = O 键的增加。使用质谱、液相色谱和明视野显微镜进行的产品分析显示反应产物的聚合状态增加，表明自由基中间体的形成之后是自由基偶联。我们的结果为漆酶氧化木质素的机制提供了进一步的见解。