Enantioselective Synthesis of l‐Homophenylalanine by Whole Cells of Recombinant Escherichia coli Expressing l‐Aminoacylase and N‐Acylamino Acid Racemase Genes from Deinococcus radiodurans BCRC12827

Abstract

L-Homophenylalanine (l-HPA) is a chiral unnatural amino acid used in the synthesis of angiotensin converting enzyme inhibitors and many pharmaceuticals. To develop a bioconversion process with dynamic resolution of N-acylamino acids for the l-HPA production, N-acylamino acid racemase (NAAAR) and l-aminoacylase (LAA) genes were cloned from Deinococcus radiodurans BCRC12827 and expressed in Escherichia coli XLIBlue. The recombinant enzymes were purified by nickel-chelate chromatography, and their biochemical properties were determined. The NAAAR had high racemization activity toward chiral N-acetyl-homophenylalanine (NAc-HPA). The LAA exhibited strict l-enantioselection to hydrolyze the NAc-l-HPA. A stirred glass vessel containing transformed E. coli cells expressing D. radiodurans NAAAR and LAA was used for the conversion of NAc-d-HPA to l-HPA. Unbalance activities of LAA and NAAAR were found in E. coli cell coexpressing laa and naaar genes, which resulted in the accumulation of an intermediate, NAc-l-HPA, in the early stage of conversion and a low productivity of 0.83 mmol l-HPA/L h. The results indicated that low activity of LAA present in the biomass is the rate-limiting factor in l-HPA production. In the case of two whole cells with separately expressed enzyme, the enzymatic activities of LAA and NAAAR could be balanced by changing the loading of individual cells. When the activities of two enzymes were fixed at 3600 U/L, 99.9% yield of l-HPA could be reached in 1 h, with a productivity of 10 mmol l-HPA/L h. The cells can be reused at least six cycles at a conversion yield of more than 96%. This is the first NAAAR/LAA process using NAc-HPA as substrate and recombinant whole cells containing Deinococcus enzymes as catalysts for the production of l-HPA to be reported.

Stereoselective synthesis of l-homophenylalanine using the carbamoylase method with in situ racemization via N-acylamino acid racemase

“…lurida DSM43134, and Amycolatopsis azurea CCRC13413 have the ability to catalyze racemization of N-acylamino acids [17,19,20]. We found that D. radiodurans NAAAR exhibited higher racemization activity to N-acetyl-HPA [9] than to N-carbamoyl-HPA. We have determined the crystal structure of D. radiodurans NAAAR and found that residues Phe 26 and Phe 30 in lid region form a van der Waals interaction to N-acetyl group of N-acetyl-Lglutamine [15].…”

“…In the previous report, we have found that the NAAAR from D. radiodurans has high racemization activity toward chiral Nacetyl-homophenylalanine [9]. The NAAAR from Amycolatopsis sp.…”

“…However, substrate (OPBA) inhibition is demonstrated in E. coli AAT activity. We had developed a novel whole-cell system, with the expression of Deinococcus radiodurans N-acylamino acid racemase (NAAAR) and L-aminoacylase genes in E. coli, which allows total conversion of racemic N-acetyl-homophenylalanine (NAc-HPA) to L-HPA with high purity [9]. Such a process however is restricted in the real application due to the low solubility of NAc-HPA (about 20 mM in 50 mM Tris-HCl buffer at pH 8.0 and 50 8C).…”

Lipases: Valuable catalysts for dynamic kinetic resolutions

“…Racemases have been combined with other enzymes, such as aminomutase, acylases and amidases, in DKR reactions to provide optically pure α-hydroxyacids, amino acids and amino acid derivatives (Asano and Yamaguchi, 2005;Baxter et al, 2012;Cox et al, 2009;Hsu et al, 2006;Resch et al, 2010;Soriano-Maldonado et al, 2014;J. Wang et al, 2013;Z.-Y.…”

Organic Solvent-Tolerant Marine Microorganisms as Catalysts for Kinetic Resolution of Cyclic β-Hydroxy Ketones

“…This process presents a broad substrate scope with no expensive cofactors, excellent stereoselectivity, environmentally benign conditions, and organic solvent-tolerance, which has resulted in its application in academic and industrial chemistry. Indeed, many microbial-derived biocatalysts, including isolated enzymes and whole cells, may serve as candidates for the kinetic resolution of cyclic β-hydroxy ketones (Baxter et al 2012;Busto et al 2010;Hsu et al 2006;Kapoor and Gupta 2012;Shakeri et al 2010;Zhang et al 2013). However, in a kinetic resolution process, the ideal biocatalyst would only convert one enantiomer to the product and leave the antipode mostly unreacted.…”