Amidohydrolase Process: Expanding the use of l-N-carbamoylase/N-succinyl-amino acid racemase tandem for the production of different optically pure l-amino acids

Soriano-Maldonado, Pablo; Rodríguez-Alonso, María José; Hernandez‐Cervantes, Carmen; Rodrı́guez-Garcı́a, Ignacio; Clemente-Jiménez, Josefa Marı́a; Rodríguez‐Vico, Felipe; Martínez‐Rodríguez, Sergio; Heras‐Vázquez, Francisco Javier Las

doi:10.1016/j.procbio.2014.04.013

Cited by 12 publications

(18 citation statements)

References 18 publications

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“…Synthesis of the hydantoins was carried out as described in [22], while that of the carbamoyls is described in [23]. The amino acids were purchased from Sigma-Adrich Química S.A. (Madrid, Spain).…”

Section: System Characterizationmentioning

confidence: 99%

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

et al. 2017

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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.

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Section: System Characterizationmentioning

confidence: 99%

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

et al. 2017

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“…Talon metal-affinity resin was purchased from Clontech Laboratories, Inc., Cloning enzymes and PCR primers were purchased from Roche Diagnostic S.L. (Barcelona, Spain) [34]. Racemic and optically pure d-and l-amino acids were purchased from Alfa Aesar GmbH & Co., K.G.…”

Section: Materials and Reagentsmentioning

confidence: 99%

“…(Barcelona, Spain). The monosubstituted hydantoins and the corresponding N-carbamoyl-amino acids used in this work were synthesized according to the potassium cyanate method for the carbamoylation and cyclation of amino acids, described elsewhere [33,34].…”

Section: Materials and Reagentsmentioning

confidence: 99%

“…l-N-carbamoylase from Geobacillus stearothermophilus CECT43 (Bslcar) and N-succinyl-amino acid racemase from G. kaustophilus CECT4264 (Gknsaar) were overexpressed and purified from Escherichia coli BL21 cells [35] harbouring the plasmids pJAVI80Rha [36] and pJPD25Rha [34], respectively, as previously described.…”

Section: Cloning Of the Genesmentioning

confidence: 99%

“…PCR amplification and subsequent cloning into the plasmid pJOE4036.1 [37] of the d-hydantoinase gene from Agrobacterium tumefaciens BQL9 (Atdhyd) and the hydantoin racemase 1 gene from A. tumefaciens C58 (AthyuA1) were performed as described in a previous work [34], with slight modifications. The PCR primers for Atdhyd gene amplification were 5 -AAAAAAACATATGGATATCATCATCAAGAACGGAAC-3 (including a NdeI site, in italics) and 5-AACTGCAGTTAATGATGATGATGATG ATGGGCGCAGGCGACGAGGGCTGG-3 (including a HindIII site, in italics, stop codon in bold and six histidine tag underlined) using pBSH1 as template [26].…”

Section: Cloning Of the Genesmentioning

confidence: 99%

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Rational re-design of the “double-racemase hydantoinase process” for optically pure production of natural and non-natural l-amino acids

Rodríguez-Alonso

Clemente-Jiménez

Rodríguez‐Vico

et al. 2015

Biochemical Engineering Journal

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Biocatalytic N‐Acylation of Amines in Water Using an Acyltransferase from Mycobacterium smegmatis

et al. 2018

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A straightforward one-step biocatalyzed synthesis of different N-acyl amides in water was accomplished using the versatile and chemoselective acyltransferase from Mycobacterium smegmatis (MsAcT). Acetylation of primary arylalkyl amines was achieved with a range of acetyl donors in biphasic systems within 1 hour and at room temperature. Vinyl acetate was the best donor which could be employed in the N-acetylation of a large range of primary amines in excellent yields (85-99%) after just 20 minutes. Other acyl donors (including formyl-, propionyl-, and butyryl-donors) were also efficiently employed in the biocatalytic N-acylation.Finally, the biocatalyst was tested in transamidation reactions using acetamide as acetyl donor in aqueous medium, reaching yields of 60-70%. This work expands the toolbox of preparative methods for the formation of N-acyl amides, describing a biocatalytic approach easy to accomplish under mild conditions in water.

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Amidohydrolase Process: Expanding the use of l-N-carbamoylase/N-succinyl-amino acid racemase tandem for the production of different optically pure l-amino acids

Cited by 12 publications

References 18 publications

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

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

Rational re-design of the “double-racemase hydantoinase process” for optically pure production of natural and non-natural l-amino acids

Biocatalytic N‐Acylation of Amines in Water Using an Acyltransferase from Mycobacterium smegmatis

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