Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain.
Department of Bio- and Environmental Sciences, TU Dresden, International Institute Zittau, Zittau, Germany.
Biotechnol Bioeng. 2021 Aug;118(8):3002-3014. doi: 10.1002/bit.27810. Epub 2021 May 14.
Fungal unspecific peroxygenases (UPOs) are efficient biocatalysts that insert oxygen atoms into nonactivated C-H bonds with high selectivity. Many oxyfunctionalization reactions catalyzed by UPOs are favored in organic solvents, a milieu in which their enzymatic activity is drastically reduced. Using as departure point the UPO secretion mutant from Agrocybe aegerita (PaDa-I variant), in the current study we have improved its activity in organic solvents by directed evolution. Mutant libraries constructed by random mutagenesis and in vivo DNA shuffling were screened in the presence of increasing concentrations of organic solvents that differed both in regard to their chemical nature and polarity. In addition, a palette of neutral mutations generated by genetic drift that improved activity in organic solvents was evaluated by site directed recombination in vivo. The final UPO variant of this evolutionary campaign carried nine mutations that enhanced its activity in the presence of 30% acetonitrile (vol/vol) up to 23-fold over PaDa-I parental type, and it was also active and stable in aqueous acetone, methanol and dimethyl sulfoxide mixtures. These mutations, which are located at the surface of the protein and in the heme channel, seemingly helped to protect UPO from harmful effects of cosolvents by modifying interactions with surrounding residues and influencing critical loops.
真菌非特异性过氧化物酶（UPO）是高效的生物催化剂，能够高选择性地将氧原子插入非活性的 C-H 键中。许多由 UPO 催化的氧化官能化反应在有机溶剂中更有利，而在有机溶剂中，其酶活性会大幅降低。本研究以阿魏侧耳（Agrocybe aegerita）的 UPO 分泌突变体（PaDa-I 变体）为出发点，通过定向进化提高了其在有机溶剂中的活性。通过随机诱变和体内 DNA 改组构建的突变文库，在不同化学性质和极性的有机溶剂存在下进行筛选。此外，还通过体内定点重组评估了由遗传漂变产生的提高有机溶剂中活性的中性突变组合。经过这一进化过程，最终的 UPO 变体携带 9 个突变，使其在 30%（体积/体积）乙腈存在下的活性提高了 23 倍，超过了 PaDa-I 亲本类型，并且在水-丙酮、甲醇和二甲基亚砜混合物中也具有活性和稳定性。这些突变位于蛋白质表面和血红素通道中，似乎通过改变与周围残基的相互作用并影响关键环，帮助 UPO 免受共溶剂的有害影响。
