Abstract

A laccase from Oudemansiella canarii was immobilized using the crosslinked enzyme aggregate (CLEA) methodology and applied in the degradation of the anthraquinonic dye Remazol Brilliant Blue R (RBBR). The immobilized laccase was superior to the free laccase in both thermal and storage stabilities. Both immobilized and free laccase decolourized 100 mg/L RBBR within 24 h at 30 °C and pH 5.0, but the former was still efficient in degrading the dye after at least 6 cycles. The relationship between the decolorization rate and the RBBR concentration obeyed Michaelis–Menten kinetics, with K_M of 0.126 ± 0.044 mmol/L and V_max of 1.412 ± 0.295 µmol/min for free laccase and K_M of 0.159 ± 0.050 mmol/L and V_max of 1.214 ± 0.242 µmol/min for immobilized laccase. Fourier transform infra-red spectroscopy (FTIR) and mass spectrometry allowed to conclude that the O. canarii laccase acts not only on the chromophore group of the dye, but that it also cleaves other covalent bonds, causing an effective fragmentation of the molecule. The Microtox assay detected a significant diminution in toxicity, a finding corroborated by the phytotoxicity test performed with lettuce seeds. Our results indicate that the immobilized laccase from O. canarii could be useful in biological strategies aiming at degrading unwanted dyes in the environment.

摘要

使用交联酶聚集体 (CLEA) 方法固定来自Oudemansiella canarii的漆酶，并应用于蒽醌染料 Remazol Brilliant Blue R (RBBR) 的降解。固定化漆酶在热稳定性和储存稳定性方面均优于游离漆酶。在 30 °C 和 pH 5.0 条件下，固定漆酶和游离漆酶均可在 24 小时内使 100 mg/L RBBR 脱色，但前者在至少 6 个循环后仍能有效降解染料。脱色率与 RBBR 浓度的关系服从 Michaelis-Menten 动力学，游离漆酶的 K _M为 0.126 ± 0.044 mmol/L，V _max为 1.412 ± 0.295 µmol/min，K _M为 0.159 ± 0.050 mmol/L 和_最大电压对于固定化漆酶，为 1.214 ± 0.242 µmol/min。傅里叶变换红外光谱 (FTIR) 和质谱分析得出结论，金丝雀漆酶不仅作用于染料的发色团，而且还切割其他共价键，导致分子有效碎裂。Microtox 检测检测到毒性显着降低，这一发现得到了用莴苣种子进行的植物毒性试验的证实。我们的结果表明，来自O. canarii的固定化漆酶可用于旨在降解环境中不需要的染料的生物学策略。