Ribitsch Doris, Herrero Acero Enrique, Przylucka Agnieszka, Zitzenbacher Sabine, Marold Annemarie, Gamerith Caroline, Tscheließnig Rupert, Jungbauer Alois, Rennhofer Harald, Lichtenegger Helga, Amenitsch Heinz, Bonazza Klaus, Kubicek Christian P, Druzhinina Irina S, Guebitz Georg M
Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria.
Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, Vienna, Tulln, Austria.
Appl Environ Microbiol. 2015 Jun;81(11):3586-92. doi: 10.1128/AEM.04111-14. Epub 2015 Mar 20.
Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased kcat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.
角质酶已显示出水解难降解合成聚合物聚对苯二甲酸乙二酯（PET）的潜力。我们之前已经表明，通过添加疏水蛋白（一种能改变表面物理化学性质的小型真菌蛋白）可以提高这种水解速率。在此，我们研究了来自嗜热栖热放线菌（Thermobifida cellulosilytica）的细菌角质酶（Thc_Cut1）与三种木霉属疏水蛋白之一（即II类疏水蛋白HFB4和HFB7以及假I类疏水蛋白HFB9b）融合后，其PET水解活性是否会进一步提高。融合酶对可溶性底物（对硝基苯乙酸和对硝基苯丁酸）的kcat值降低，玻璃的亲水性大幅下降，但PET的疏水性仅发生微小变化。当该酶与HFB4或HFB7融合时，PET的水解比游离酶水平提高了16倍以上，而酶与疏水蛋白的混合物最多仅导致4倍的增加。与非II类疏水蛋白HFB9b融合并没有比酶 - 疏水蛋白混合物提高水解速率，但在酶处理前将PET与疏水蛋白预孵育时，HFB9b表现最佳。随着产物单（2 - 羟乙基）对苯二甲酸酯相对于主要产物对苯二甲酸的浓度增加，融合酶的水解模式与Thc_Cut1不同。小角X射线散射（SAXS）分析表明，融合蛋白的散射对比度高于游离蛋白，表明其构象发生变化或蛋白质聚集增强。我们的数据表明，角质酶对PET的水解水平通过与疏水蛋白融合可显著提高。数据进一步表明，这可能涉及疏水蛋白与角质酶的结合及其活性中心构象的变化。
