Rational re-design of the 'double-racemase hydantoinase process' for optically pure production of natural and non-natural L-amino acids

摘要

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 degrees C, with the maximum reaction rate at 60 degrees 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. (C) 2015 Elsevier B.V. All rights reserved.