School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin 4, Ireland.
Institute for Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, Belgrade, 11000, Serbia.
Appl Microbiol Biotechnol. 2018 Jun;102(12):5121-5131. doi: 10.1007/s00253-018-8994-5. Epub 2018 Apr 24.
We evaluated the kinetic characteristics of wild type (WT) and three engineered variants (RVC10, RV145, and C10_N322S) of tyrosinase from Ralstonia solanacearum and their potential as biocatalysts to produce halogenated catechols. RV145 exhibited a 3.6- to 14.5-fold improvement in catalytic efficiency (k/K) with both reductions in K and increases in k compared to WT, making it the best R. solanacearum tyrosinase variant towards halogenated phenols. RVC10 also exhibited increases in catalytic efficiency with all the tested phenols. A single-mutation variant (C10_N322S) exhibited the greatest improvement in k but lowest improvement in catalytic efficiency due to an increase in K compared to WT. Consistent with kinetic characteristics, biotransformation experiments showed that RV145 was a superior biocatalyst in comparison to WT. To prevent through conversion of the catechol to quinone, ascorbic acid (AA) was added to the biotransformation medium in 1:2 (substrate:AA) ratio resulting in a catechol yield of > 90%. Flask experiments with 10 mM 4-iodophenol and 10 μg/mL of the RV145 enzyme yielded 9.5 mM 4-iodocatechol in the presence of 20 mM AA in 30 min. Similarly, 10 mM 4-fluorophenol was completely consumed by 20 μg/mL of RV145 enzyme and yielded 9.2 mM 4-fluorocatechol in the presence of 20 mM AA in 80 min. The biotransformation of 20 mM 4-fluorphenol was incomplete (93%) and the yield of 4-flurocatechol was 87.5%. The 4-halophenol conversion rates and product yields obtained in this study are the highest reported using tyrosinase or any other enzyme.
我们评估了野型（WT）和三种工程变体（RVC10、RV145 和 C10_N322S）的酪氨酸酶的动力学特性，以及它们作为生物催化剂生产卤代儿茶酚的潜力。与 WT 相比，RV145 对卤代酚的催化效率（k/K）提高了 3.6-14.5 倍，同时降低了 K 值，提高了 k 值，使其成为最好的罗尔斯通氏菌酪氨酸酶变体。RVC10 对所有测试的酚类化合物的催化效率也有所提高。与 WT 相比，单一突变变体（C10_N322S）的 k 值提高最大，但由于 K 值增加，催化效率提高最低。与动力学特性一致，生物转化实验表明，与 WT 相比，RV145 是一种更优越的生物催化剂。为了防止儿茶酚转化为醌，在生物转化培养基中以 1:2（底物：AA）的比例添加抗坏血酸（AA），导致儿茶酚的产率>90%。在含有 20 mM AA 的情况下，10 mM 4-碘苯酚和 10 μg/mL RV145 酶的摇瓶实验在 30 分钟内产生 9.5 mM 4-碘儿茶酚。同样，在含有 20 mM AA 的情况下，20 μg/mL RV145 酶完全消耗 10 mM 4-氟苯酚，并在 80 分钟内生成 9.2 mM 4-氟儿茶酚。20 mM 4-氟苯酚的生物转化不完全（93%），4-氟儿茶酚的产率为 87.5%。本研究中获得的 4-卤代苯酚转化率和产物收率是使用酪氨酸酶或任何其他酶获得的最高值。
