l-Amino Acid Production by a Immobilized Double-Racemase Hydantoinase Process: Improvement and Comparison with a Free Protein System

Abstract

Protein immobilization is proving to be an environmentally friendly strategy for manufacturing biochemicals at high yields and low production costs. This work describes the optimization of the so-called "double-racemase hydantoinase process," a system of four enzymes used to produce optically pure L-amino acids from a racemic mixture of hydantoins. The four proteins were immobilized separately, and, based on their specific activity, the optimal whole relation was determined. The first enzyme, D,L-hydantoinase, preferably hydrolyzes D-hydantoins from D,L-hydantoins to N-carbamoyl-D-amino acids. The remaining L-hydantoins are racemized by the second enzyme, hydantoin racemase, and continue supplying substrate D-hydantoins to the first enzyme. N-carbamoyl-D-amino acid is racemized in turn to N-carbamoyl-L-amino acid by the third enzyme, carbamoyl racemase. Finally, the N-carbamoyl-L-amino acid is transformed to L-amino acid by the fourth enzyme, L-carbamoylase. Therefore, the product of one enzyme is the substrate of another. Perfect coordination of the four activities is necessary to avoid the accumulation of reaction intermediates and to achieve an adequate rate for commercial purposes. The system has shown a broad pH optimum of 7-9, with a maximum activity at 8 and an optimal temperature of 60 • C. Comparison of the immobilized system with the free protein system showed that the reaction velocity increased for the production of norvaline, norleucine, ABA, and homophenylalanine, while it decreased for L-valine and remained unchanged for L-methionine.

N-succinylamino acid racemases: Enzymatic properties and biotechnological applications

“…Some efforts have also been carried out to study the immobilization of NSARs [63,67,88,89]. Enzyme immobilization resulted in a 75% loss of GkaNSAR activity [67] and 90 % with immobilized DraNSAR [63] when compared to the enzymes in solution.…”

Overview on Multienzymatic Cascades for the Production of Non-canonical α-Amino Acids

“…(D) “Double-racemase Hydantoinase Process” for the production of enantiopure L -α-AAs. Different combinations of purified and immobilized D -hydantoinase (DHyd) and hydantoin racemase (HR) from Agrobacterium species, N -succinyl-amino acid racemase (NSAR) from Geobacillus kaustophilus and L -carbamoylase (LCar) from Geobacillus stearothermopillus allowed the production of different enantiopure NcAAs; L -Met and L -Val were also efficiently produced with this system ( Rodríguez-Alonso et al, 2015 , 2016 , 2017 ).…”

“…Engineered dihydropyrimidinase from Brevibacillus agri (L159V mutant), Bacillus kaustophilus L -carbamoylase and Deinococcus radiodurans NSAR were purified, and allowed the production of L -homophenylalanine (90% yield, 5 h, Lo et al, 2009 ). This L -enantiospecific MEC system has further been expanded by inclusion of a hydantoin racemase, speeding up the process by racemization of the remaining L -5-monosubstituted hydantoin for substrates whose chemical racemization is not favored ( Rodríguez-Alonso et al, 2015 , 2016 , 2017 ; Figure 1D ). Using purified and immobilized enzymes, the proposed “double-racemase Hydantoinase Process” was efficiently applied for the synthesis of different NcAAs ( L -norvaline, L -norleucine, L -ABA, L -homophenylalanine).…”

“…Using purified and immobilized enzymes, the proposed “double-racemase Hydantoinase Process” was efficiently applied for the synthesis of different NcAAs ( L -norvaline, L -norleucine, L -ABA, L -homophenylalanine). The immobilized system could be reused 15 times, retaining 80% of the initial activity ( Rodríguez-Alonso et al, 2017 ).…”

Biocatalysis and Biotransformations

“…The synthesis of S-adenosylmethionine was investigated by Niu and coworkers using methionine adenosyltransferases [20]. L-amino acid production was investigated by Rodríguez-Alonso and coworkers who used double enzymatic systems based on the application of racemases and hydantoinases to produce norvaline, norleucine, and homophenylalanine [21]. The conversion of furans, including 5-(hydroxymethyl) furfural (HMF) and furfural, by Bayer-Villiger monooxygenases was investigated by Kumar and Fraaije [22].…”