Hydantoinase Process Research Articles

Amino Acids: Unnaturally, Naturally! Hydantoinase Process for Manufacturing L‐ and D‐Amino Acids

AbstractFor Abstract see ChemInform Abstract in Full Text.

Molecular Cloning and Biochemical Characterization of L-N-Carbamoylase from Sinorhizobium meliloti CECT4114

An N-carbamoyl-L-amino acid amidohydrolase (L-N-carbamoylase) from Sinorhizobium meliloti CECT 4114 was cloned and expressed in Escherichia coli. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl α-amino acid to the corresponding free amino acid, and its purification has shown it to be strictly L-specific. The enzyme showed broad substrate specificity, and it is the first L-N-carbamoylase that hydrolyses N-carbamoyl-L-tryptophan as well as N-carbamoyl L-amino acids with aliphatic substituents. The apparent K_m values for N-carbamoyl-L-methionine and tryptophan were very similar (0.65 ± 0.09 and 0.69 ± 0.08 mM, respectively), although the rate constant was clearly higher for the L-methionine precursor (14.46 ± 0.30 s^–1) than the L-tryptophan one (0.15 ± 0.01 s^–1). The enzyme also hydrolyzed N-formyl-L-methionine (k_cat/K_m = 7.10 ± 2.52 s^–1·mM^–1) and N-acetyl-L-methionine (k_cat/K_m = 12.16 ± 1.93 s^–1·mM^–1), but the rate of hydrolysis was lower than for N-carbamoyl-L-methionine (k_cat/K_m = 21.09 ± 2.85). This is the first L-N-carbamoylase involved in the ‘hydantoinase process’ that has hydrolyzed N-carbamoyl-L-cysteine, though less efficiently than N-carbamoyl-L-methionine. The enzyme did not hydrolyze ureidosuccinic acid or 3-ureidopropionic acid. The native form of the enzyme was a homodimer with a molecular mass of 90 kDa. The optimum conditions for the enzyme were 60°C and pH 8.0. Enzyme activity required the presence of divalent metal ions such as Ni²⁺, Mn²⁺, Co²⁺ and Fe²⁺, and five amino acids putatively involved in the metal binding were found in the amino acid sequence.

Enzymatic synthesis of enantiomerically enriched d- and l-3-silylated alanines by deracemization of dl-5-silylmethylated hydantoins

The hydantoinase process was shown to be extendable to the production of highly lipophilic, silicon-containing amino acids. Two hydantoinases of different origin and stereoselectivities and one l- N-carbamoylase were used for the highly stereoselective bioconversion of (dimethyl)phenylsilyl- and 1-methyl-1-silacyclopentyl substituted alanine derivatives. The enantiomeric purities and absolute configuration of the products were determined with reference compounds that were synthesized with the aid of the Evans oxazolidinone auxiliary.

Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine.

Using directed evolution, we have improved the hydantoinase process for production of L-methionine (L-met) in Escherichia coli. This was accomplished by inverting the enantioselectivity and increasing the total activity of a key enzyme in a whole-cell catalyst. The selectivity of all known hydantoinases for D-5-(2-methylthioethyl)hydantoin (D-MTEH) over the L-enantiomer leads to the accumulation of intermediates and reduced productivity for the L-amino acid. We used random mutagenesis, saturation mutagenesis, and screening to convert the D-selective hydantoinase from Arthrobacter sp. DSM 9771 into an L-selective enzyme and increased its total activity fivefold. Whole E. coli cells expressing the evolved L-hydantoinase, an L-N-carbamoylase, and a hydantoin racemase produced 91 mM L-met from 100 mM D,L-MTEH in less than 2 h. The improved hydantoinase increased productivity fivefold for >90% conversion of the substrate. The accumulation of the unwanted intermediate D-carbamoyl-methionine was reduced fourfold compared to cells with the wild-type pathway. Highly D-selective hydantoinase mutants were also discovered. Enantioselective enzymes rapidly optimized by directed evolution and introduced into multienzyme pathways may lead to improved whole-cell catalysts for efficient production of chiral compounds.

ChemInform Abstract: Enzymatic Processes for L‐Phenylalanine Production

With increasing demand for L-Phenylalanine, the enzymatic processes for the production of this amino acid have developed remarkably over the past several years. Among these processes, the followings have reached their technical levels for the commercial production : transaminase process with phenylpyruvic acid, phenylalanine dehydrogenase process with phenylpyruvic acid, phenylalanine ammonia-lyase process with trans -cinnamic acid and hydantoinase process with D, L-benzylhydantoin as substrate, respectively. Acetamidocinnamic acid, D, L-phenylalanine amide and D, L-phenyllactic acid are also useful as the substrate for enzymatic production of L-phenylalanine.

酵素法によるＬ‐フェニルアラニンの生産

With increasing demand for L-Phenylalanine, the enzymatic processes for the production of this amino acid have developed remarkably over the past several years. Among these processes, the followings have reached their technical levels for the commercial production : transaminase process with phenylpyruvic acid, phenylalanine dehydrogenase process with phenylpyruvic acid, phenylalanine ammonia-lyase process with trans -cinnamic acid and hydantoinase process with D, L-benzylhydantoin as substrate, respectively. Acetamidocinnamic acid, D, L-phenylalanine amide and D, L-phenyllactic acid are also useful as the substrate for enzymatic production of L-phenylalanine.