Enzymatic dynamic kinetic resolution of racemic N-formyl- and N-carbamoyl-amino acids using immobilized l-N-carbamoylase and N-succinyl-amino acid racemase

Abstract

Taking advantage of the catalytic promiscuity of L-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) and N-succinyl-amino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR), we have evaluated the production of different optically pure L-α-amino acids starting from different racemic N-formyl- and N-carbamoyl-amino acids using a dynamic kinetic resolution approach. The enzymes were immobilized on two different solid supports, resulting in improved stability of the enzymes in terms of thermostability and storage when compared to the enzymes in solution. The bienzymatic system retained up to 80% conversion efficiency after 20 weeks at 4 °C and up to 90% after 1 week at 45 °C. The immobilization process also resulted in a great enhancement of the activity of BsLcar toward N-formyl-tryptophan, showing for the first time that substrate specificity of L-carbamoylases can be influenced by this approach. The system was effective for the biosynthesis of natural and unnatural L-amino acids (enantiomeric excess (e.e.) >99.5%), such as L-methionine, L-alanine, L-tryptophan, L-homophenylalanine, L-aminobutyric acid, and L-norleucine, with a higher performance toward N-formyl-α-amino acid substrates. Biocatalyst reuse was studied, and after 10 reaction cycles, over 75% activity remained.

Toward a cell-free hydantoinase process: screening for expression optimization and one-step purification as well as immobilization of hydantoinase and carbamoylase

“…However, immobilization of the l -carbamoylase from Bacillus kaustophilus on Eupergit C was investigated and a lower optimal specific activity of 2.91 U/mg was achieved (Yen et al 2010). Immobilization of the l -carbamoylase from Geobacillus stearothermophilus CECT43 on Sepabeads EC-HFA/S was reported to result in a maximum specific activity of around 14.00 U/mg by Soriano-Maldonado et al (2014) which is comparable to our results.…”

Immobilization of a multi‐enzyme system for L‐amino acids production

“…Analyzed separately, the enzymes give conversions of 80–95% on the best support for each one, but the cascade did not yield more than 64.1% and fell to 44.7% over the cycles. There are few examples of immobilized enzymatic cascades; bi‐enzymatic systems have been described by Ragnitz et al , 2001; Yen et al , 2011 and Soriano‐Maldonado et al , 2015 . The results of the first two were not very promising, while those of the third study describe a scalable system with high yields and e.e.>99.5%.…”

“…Few research works refer to a multi‐step reaction with immobilized proteins and this study may therefore be a promising incentive for industrial applications in biocatalysis . An enzymatic cascade implies continuous substrate feedback and free mass transfer.…”

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

“…This increase in the optimum temperature is an advantage when the substrates are poorly soluble and a slight increase in temperature is necessary to facilitate their dissolution. L-N-carbamoylase and hydantoinase from Arthrobacter aurescens showed similar behavior when immobilized in EAH-Sepharose 4B, presenting a broad spectrum of pH and remaining active at higher temperatures, from 25 to 60 °C and 25 to 45 °C, respectively [20].…”

“…A bi-enzymatic system consisting of L-N-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) and N-succinyl-amino acid racemase from Geobacillus kaustophilus CECT 4264 (GkNSAAR) as carbamoyl racemase was immobilized in Immobeads IB-161, showing 75% activity after 10 reaction cycles. For both enzymes the specific activity decreased when they were immobilized, but even with this setback, the balance was positive, due to the greater stability and pH range, the greater number of substrates able to hydrolyze, and the increased reusage capacity of the system [20].…”