Rational re-design of the 'double-racemase hydantoinase process' for optically pure production of natural and non-natural l-amino acids
| Autor: | María José Rodríguez-Alonso, Josefa María Clemente-Jiménez, Felipe Rodríguez-Vico, Francisco Javier Las Heras Vázquez |
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| Rok vydání: | 2015 |
| Předmět: |
chemistry.chemical_classification
Environmental Engineering biology Stereochemistry Biomedical Engineering Substrate (chemistry) Bioengineering Agrobacterium tumefaciens biology.organism_classification Catalysis Kinetic resolution Amino acid Hydantoin racemase Enzyme Cascade reaction chemistry Biotechnology |
| Zdroj: | Biochemical Engineering Journal. 101:68-76 |
| ISSN: | 1369-703X |
| DOI: | 10.1016/j.bej.2015.05.003 |
| Popis: | The “hydantoinase process” is a well-established method for the industrial production of optically pure d -amino acids. However, due to the strict d -enantioselectivity of most hydantoinase enzymes, the process is less efficient for l -amino acid production. We present a new chemo-enzymatic cascade reaction for natural and non-natural l -amino acid production from racemic mixtures of 5-monosubstituted hydantoins. This system comprised the following enzymes: d -hydantoinase from Agrobacterium tumefaciens BQL9, hydantoin racemase 1 from A. tumefaciens C58 and l -N-carbamoylase from Geobacillus stearothermophilus CECT43, together with N-succinyl-amino acid racemase from G. kaustophilus CECT4264. This latter presents catalytic promiscuity and racemizes N-carbamoyl-amino acids. This activity avoids the accumulation of N-carbamoyl- d -amino acid in the reaction due to the strict d -enantioselectivity of the hydantoinase. The optimum pH for the system proved to be 8.0, whereas optimum temperature range was 50–65 °C, with the maximum reaction rate at 60 °C. The metal ion cobalt was added directly to the reaction mixture (end concentration 1 mM), but in the case of d -hydantoinase, overexpression in presence of 0.5 mM Co2+ was also necessary. The enzymatic cascade reaction produced different optically pure l -amino acids by dynamic kinetic resolution, achieving 100% conversion even at high substrate concentrations (100 mM) with no noticeable inhibition. This total conversion demonstrates that the “double-racemase hydantoinase process” upgrades the classical “hydantoinase process” for natural and non-natural l -amino acid production. |
| Databáze: | OpenAIRE |
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