Development of Dynamic Kinetic Resolution Processes for Biocatalytic Production of Natural and Nonnatural l-Amino Acids

Abstract

Two different dynamic kinetic resolution processes for the production of a number of natural and nonnatural l-amino acids at 100% chemical and optical yield have recently been established at Degussa. The first process is based on the dynamic kinetic resolution of 5-monosubstituted hydantoins using tailor-made whole-cell biocatalysts coexpressing a l-carbamoylase, a hydantoin racemase, and a hydantoinase. The hydantoin-converting pathway was optimized by adjusting expression levels of the respective enzymes as well as by inverting the enantioselectivity of the d-selective hydantoinase. This resulted overall in a 50-fold improved productivity and significant reduction of biocatalyst cost. The second process is based on the dynamic kinetic resolution of N-acetyl amino acids using an acylase in combination with a novel racemase from Amycolatopsis orientalis subsp. lurida. This racemase could overcome the problem of substrate inhibition and requirement of high concentrations of divalent metal ions which limits the use of other N-acylamino acid racemases described in the literature.

Enzymatic Racemisation and its Application to Synthetic Biotransformations

“…lurida and an amino acylase. [92] Currently, several hundred tons of l-methionine are produced annually by kinetic resolution in an enzyme membrane reactor. [93] Previously, kinetic resolution of N-acetyl-dl-amino acids was achieved by using an enantiospecific l-amino acylase from Aspergillus oryzae which hydrolyses only the lenantiomer to produce a mixture of the corresponding l-amino acid and unreacted N-acetyl-d-amino acid.…”

“…[99] Further examples of substrates for hydantoin racemases from Arthrobacter spp. DSM 9771 [92] and DSM 3745 [100] are depicted in Scheme 15.…”

“…[103] Today, it is commercially applied at a scale of >1000 tons per year for the above-mentioned amino acids which are used as side chains for b-lactam antibiotics, such as ampicillin and amoxicillin, respectively. It was only recently that the hydantoin process became also feasible on a large scale for l-amino acids due to improvement of the productivity by using a recombinant whole-cell biocatalyst, [92] which allows commercialisation even for low-priced amino acids such as l-methionine.…”

Molecular Cloning and Biochemical Characterization of <i>L</i>-N-Carbamoylase from <i>Sinorhizobium meliloti</i> CECT4114

“…However, L -N-carbamoylases that hydrolyze N-carbamoyl-L -tryptophan as well as N-carbamoyl L -amino acids with aliphatic substituents have not been reported [Hu et al, 2003]. This broad substrate specifi c L -N-carbamoylase could increase the commercial application of the 'hydantoinase process' currently used for obtaining D -amino acids, allowing L -amino acids to also be obtained [May et al, 2002]. The possibility of reducing production costs by using recombinant cells of Escherichia coli , thus increasing activity by overexpression of the enzymes involved in this reaction could constitute a commercially feasible process for L -amino acid production.…”

Multi‐Enzymatic Cascade Reactions: Overview and Perspectives

“…[161] A great potential of multi-step synthesis is in the process for N-acetyl-d-neuraminic acid (Neu5Ac), a starting material for anti-influenza virus agents that is of great commercial value. Neu5Ac can be obtained in a onepot reaction catalyzed by epimerases and aldolases even with different techniques for enzyme immobilization which makes the multi-enzyme process very attractive for large-scale production.…”