Biocatalytic versatility of engineered and wild-type tyrosinase from R-solanacearum for the synthesis of 4-halocatechols
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Davis, ReetaMolloy, Susan
Quigley, Blathnaid
Nikodinović-Runić, Jasmina
Solano, Francisco
O'Connor, Kevin
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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 (cat)/K (m)) with both reductions in K (m) and increases in k (cat) 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 (cat) but lowest improvement in catalytic efficiency due to an increase in K (m) 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 (subs...trate:AA) ratio resulting in a catechol yield of gt 90%. Flask experiments with 10 mM 4-iodophenol and 10 mu g/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 mu g/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.
Keywords:
Tyrosinase / Enzyme engineering / Enzyme catalysis / 4-Halophenol / 4-HalocatecholSource:
Applied Microbiology and Biotechnology, 2018, 102, 12, 5121-5131Publisher:
- Springer, New York
DOI: 10.1007/s00253-018-8994-5
ISSN: 0175-7598
PubMed: 29691629
WoS: 000432692300011
Scopus: 2-s2.0-85045904455
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Institut za molekularnu genetiku i genetičko inženjerstvoTY - JOUR AU - Davis, Reeta AU - Molloy, Susan AU - Quigley, Blathnaid AU - Nikodinović-Runić, Jasmina AU - Solano, Francisco AU - O'Connor, Kevin PY - 2018 UR - https://imagine.imgge.bg.ac.rs/handle/123456789/1175 AB - 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 (cat)/K (m)) with both reductions in K (m) and increases in k (cat) 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 (cat) but lowest improvement in catalytic efficiency due to an increase in K (m) 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 gt 90%. Flask experiments with 10 mM 4-iodophenol and 10 mu g/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 mu g/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. PB - Springer, New York T2 - Applied Microbiology and Biotechnology T1 - Biocatalytic versatility of engineered and wild-type tyrosinase from R-solanacearum for the synthesis of 4-halocatechols EP - 5131 IS - 12 SP - 5121 VL - 102 DO - 10.1007/s00253-018-8994-5 ER -
@article{ author = "Davis, Reeta and Molloy, Susan and Quigley, Blathnaid and Nikodinović-Runić, Jasmina and Solano, Francisco and O'Connor, Kevin ", year = "2018", abstract = "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 (cat)/K (m)) with both reductions in K (m) and increases in k (cat) 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 (cat) but lowest improvement in catalytic efficiency due to an increase in K (m) 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 gt 90%. Flask experiments with 10 mM 4-iodophenol and 10 mu g/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 mu g/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.", publisher = "Springer, New York", journal = "Applied Microbiology and Biotechnology", title = "Biocatalytic versatility of engineered and wild-type tyrosinase from R-solanacearum for the synthesis of 4-halocatechols", pages = "5131-5121", number = "12", volume = "102", doi = "10.1007/s00253-018-8994-5" }
Davis, R., Molloy, S., Quigley, B., Nikodinović-Runić, J., Solano, F.,& O'Connor, K.. (2018). Biocatalytic versatility of engineered and wild-type tyrosinase from R-solanacearum for the synthesis of 4-halocatechols. in Applied Microbiology and Biotechnology Springer, New York., 102(12), 5121-5131. https://doi.org/10.1007/s00253-018-8994-5
Davis R, Molloy S, Quigley B, Nikodinović-Runić J, Solano F, O'Connor K. Biocatalytic versatility of engineered and wild-type tyrosinase from R-solanacearum for the synthesis of 4-halocatechols. in Applied Microbiology and Biotechnology. 2018;102(12):5121-5131. doi:10.1007/s00253-018-8994-5 .
Davis, Reeta, Molloy, Susan, Quigley, Blathnaid, Nikodinović-Runić, Jasmina, Solano, Francisco, O'Connor, Kevin , "Biocatalytic versatility of engineered and wild-type tyrosinase from R-solanacearum for the synthesis of 4-halocatechols" in Applied Microbiology and Biotechnology, 102, no. 12 (2018):5121-5131, https://doi.org/10.1007/s00253-018-8994-5 . .