Low-expression levels remain a challenge in the quest to use the small laccase (rSLAC) as a viable catalyst. In this study, a recombinant Pichia pastoris strain (rSLAC-GAP-AOX) producing rSLAC under both AOX and GAP promoters (located in two different plasmids) was generated and cultivated in the presence of methanol and mixed feed (methanol:glycerol). Induction with methanol resulted in a maximum laccase activity of 1200 U/L for rSLAC-GAP-AOX which was approximately 2.4-fold higher than rSLAC-AOX and 5.1-fold higher than rSLAC-GAP. The addition of methanol:glycerol in a stoichiometric ratio of 9:1 consistently improved biomass and led to a 1.5-fold increase in rSLAC production as compared to induction with methanol alone. The rSLAC removed 95% of 5 mg/L ciprofloxacin (CIP) and 99% of 100 mg/L tetracycline (TC) in the presence of a mediator. Removal of TC resulted in complete elimination of antibacterial activity while up to 48% reduction in antibacterial activity was observed when CIP was removed. Overall, the present study highlights the effectiveness of a double promoter system in enhancing SLAC production.

在寻求使用小漆酶 (rSLAC) 作为可行催化剂的过程中，低表达水平仍然是一个挑战。在这项研究中，重组毕赤酵母在 AOX 和 GAP 启动子（位于两个不同的质粒中）下产生 rSLAC 的菌株（rSLAC-GAP-AOX）在甲醇和混合饲料（甲醇：甘油）存在下产生和培养。甲醇诱导导致 rSLAC-GAP-AOX 的最大漆酶活性为 1200 U/L，比 rSLAC-AOX 高约 2.4 倍，比 rSLAC-GAP 高 5.1 倍。与单独用甲醇诱导相比，以 9:1 的化学计量比添加甲醇:甘油持续改善生物量，并导致 rSLAC 产量增加 1.5 倍。在介质存在的情况下，rSLAC 去除了 95% 的 5 mg/L 环丙沙星 (CIP) 和 99% 的 100 mg/L 四环素 (TC)。去除 TC 导致抗菌活性完全消除，而当去除 CIP 时，观察到抗菌活性降低高达 48%。