University of Belgrade-Faculty of Chemistry, Studentski Trg 12-16, 11000 Belgrade, Serbia.
Purdue Institute of Inflammation, Immunology and Infectious Disease, Molecular Evolution, Protein Engineering and Production, Purdue University, 207 S. Martin Jischke Dr., West Lafayette, IN 47907, USA; Institute of Molecular Biotechnology, RWTH Aachen University Worringerweg 1, 52074 Aachen, Germany.
Enzyme Microb Technol. 2020 May;136:109509. doi: 10.1016/j.enzmictec.2020.109509. Epub 2020 Jan 15.
Azo dyes are toxic and carcinogenic synthetic pigments that accumulate as pollutants in aquatic bodies near textile factories. The pigments are structurally diverse, and bioremediation is mostly limited to single dye compounds or related groups. Versatile peroxidase (VP) from Pleurotus eryngii is a heme-containing peroxidase with a broad substrate spectrum that can break down many structurally distinct pollutants, including azo dyes. The utilization of this enzyme could be facilitated by engineering to modify its catalytic activity and substrate range. We used saturation mutagenesis to alter two amino acids in the catalytic tryptophan environment of VP (V160 and A260). Library screening with three azo dyes revealed that these two positions had a significant influence on substrate specificity. We were able to isolate and sequence VP variants with up to 16-fold higher catalytic efficiency for different azo dyes. The same approach could be used to select for VP variants that catalyze the degradation of many other types of pollutants. To allow multiple cycles of dye degradation, we immobilized VP on the surface of yeast cells and used washed cell wall fragments after lysis. VP embedded in the cell wall retained ∼70 % of its initial activity after 10 cycles of dye degradation each lasting 12 h, making this platform ideal for the bioremediation of environments contaminated with azo dyes.
偶氮染料是有毒和致癌的合成颜料，它们作为污染物在靠近纺织厂的水体中积累。这些颜料在结构上具有多样性，生物修复主要限于单一染料化合物或相关基团。来自草菇（Pleurotus eryngii）的多功能过氧化物酶（VP）是一种含有血红素的过氧化物酶，具有广泛的底物谱，可以分解许多结构上不同的污染物，包括偶氮染料。通过工程改造来修饰其催化活性和底物范围，可以促进该酶的利用。我们使用饱和诱变改变了 VP 催化色氨酸环境中的两个氨基酸（V160 和 A260）。用三种偶氮染料进行文库筛选表明，这两个位置对底物特异性有显著影响。我们能够分离和测序 VP 变体，它们对不同偶氮染料的催化效率提高了高达 16 倍。同样的方法可以用于选择催化许多其他类型污染物降解的 VP 变体。为了允许多次染料降解循环，我们将 VP 固定在酵母细胞表面，并在裂解后使用洗涤过的细胞壁碎片。嵌入细胞壁中的 VP 在 10 次每次持续 12 小时的染料降解循环后保留了初始活性的约 70%，使得该平台非常适合受偶氮染料污染的环境的生物修复。
