Recombinant polycistronic structure of hydantoinase process genes in Escherichia coli for the production of optically pure D-amino acids

Two recombinant reaction systems for the production of optically pure D-amino acids from different D,L-5-monosubstituted hydantoins were constructed. Each system contained three enzymes, two of which were D-hydantoinase and D-carbamoylase from Agrobacterium tumefaciens BQL9. The third enzyme was hydantoin racemase 1 for the first system and hydantoin racemase 2 for the second system, both from A. tumefaciens C58. Each system was formed by using a recombinant Escherichia coli strain with one plasmid harboring three genes coexpressed with one promoter in a polycistronic structure. The D-carbamoylase gene was cloned closest to the promoter in order to obtain the highest level of synthesis of the enzyme, thus avoiding intermediate accumulation, which decreases the reaction rate. Both systems were able to produce 100% conversion and 100% optically pure D-methionine, D-leucine, D-norleucine, D-norvaline, D-aminobutyric acid, D-valine, D-phenylalanine, D-tyrosine, and D-tryptophan from the corresponding hydantoin racemic mixture. For the production of almost all D-amino acids studied in this work, system 1 hydrolyzed the 5-monosubstituted hydantoins faster than system 2.     

Overexpression and characterization of hydantoin racemase from Agrobacterium tumefaciens C58

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific D-carbamoylase guarantee the total conversion from D,L-5-monosubstituted hydantoins with a low velocity of racemization to optically pure D-amino acids. In this work we have cloned and expressed the hydantoin racemase gene from two strains of Agrobacterium tumefaciens, C58 and LBA4404, in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure by using immobilized cobalt affinity chromatography and showed an apparent molecular mass of 32,000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 100,000 Da, suggesting that the native enzyme is a tetramer. The optimal conditions for hydantoin racemase activity were pH 7.5 and 55 degrees C with L-5-ethylhydantoin as substrate. Enzyme activity was slightly affected by the addition of Ni(2+) and Co(2+) and strongly inhibited by Cu(2+) and Hg(2+). No effect on enzyme activity was detected with Mn(2+), EDTA, or DTT. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.     

Cloning and characterization of genes from Agrobacterium sp. IP I-671 involved in hydantoin degradation

Cloning and sequencing of a 7.1 kb DNA fragment from Agrobacterium sp IP I-671 revealed seven open reading frames (ORFs) encoding D-hydantoinase, D-carbamoylase and putative hydantoin racemase, D-amino acid oxidase and NAD(P)H-flavin oxidoreductase. Two incomplete ORFs flanking the hydantoin utilization genes showed similarities to genes involved in transposition. Expression of the D-hydantoinase and D-carbamoylase gene in Escherichia coli gave mainly inactive protein concentrated in inclusion bodies, whereas homologous expression on an RSF1010 derivative increased hydantoinase and D-carbamoylase activity 2.5-fold and 10-fold, respectively, in this strain. Inactivation of the D-carbamoylase gene in Agrobacterium sp IP I-671 led to a complete loss of detectable carbamoylase activity whereas the low hydantoinase activity remaining after inactivation of the D-hydantoinase gene indicated the presence of a second hydantoinase-encoding gene. Two plasmids of 80 kb and 190 kb in size were identified by pulsed-field gel electrophoresis and the cloned hydantoin utilization genes were found to be localized on the 190 kb plasmid.     

Biochemical characterization of a novel hydantoin racemase from Agrobacterium tumefaciens C58

A novel hydantoin racemase gene of Agrobacterium tumefaciens C58 (AthyuA2) has been cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure and showed an apparent molecular mass of 27000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of approximately 100000 Da, suggesting that the native enzyme is a tetramer. The optimum pH and temperature for hydantoin racemase activity were 7.5 and 55 degrees C, respectively, with L-5-ethylhydantoin as substrate. Enzyme activity was strongly inhibited by Cu(2+) and Hg(2+). No effect on enzyme activity was detected with any other divalent cations, EDTA or DTT, suggesting that it is not a metalloenzyme. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.     

Binding studies of hydantoin racemase from Sinorhizobium meliloti by calorimetric and fluorescence analysis

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific d-carbamoylase guarantee the total conversion from d,l-5-monosubstituted hydantoins with a low velocity of racemization, to optically pure d-amino acids. Hydantoin racemase from Sinorhizobium meliloti was expressed in Escherichia coli. Calorimetric and fluorescence experiments were then carried out to obtain the thermodynamic binding parameters, deltaG, deltaH and DeltaS for the inhibitors L- and D-5-methylthioethyl-hydantoin. The number of active sites is four per enzyme molecule (one per monomer), and the binding of the inhibitor is entropically and enthalpically favoured under the experimental conditions studied. In order to obtain information about amino acids involved in the active site, four different mutants were developed in which cysteines 76 and 181 were mutated to Alanine and Serine. Their behaviour shows that these cysteines are essential for enzyme activity, but only cysteine 76 affects the binding to these inhibitors.     

Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization

In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-amino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoylase and a hydantoin racemase. The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described L-N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia coli and the recombinant protein purified to homogeneity and further characterized. The optimal condition for racemase activity were pH 8.5 and 55 degrees C with L-5-benzylhydantoin as substrate. The enzyme was completely inhibited by HgCL2 and iodoacetamide and stimulated by addition of dithiothreitol. No effect on enzyme activity was seen with EDTA. The enzyme showed preference for hydantoins with arylalkyl side chains. Kinetic studies revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhydantoin. Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.     

Site-directed mutagenesis indicates an important role of cysteines 76 and 181 in the catalysis of hydantoin racemase from Sinorhizobium meliloti

Hydantoin racemase enzyme plays a crucial role in the reaction cascade known as "hydantoinase process." In conjunction with a stereoselective hydantoinase and a stereospecific carbamoylase, it allows the total conversion from D,L-5-monosubstituted hydantoins, with a low rate of racemization, to optically pure D- or L-amino acids. Residues Cys76 and Cys181 belonging to hydantoin racemase from Sinorhizobium meliloti (SmeHyuA) have been proved to be involved in catalysis. Here, we report biophysical data of SmeHyuA Cys76 and Cys181 to alanine mutants, which point toward a two-base mechanism for the racemization of 5-monosubstituted hydantoins. The secondary and the tertiary structure of the mutants were not significantly affected, as shown by circular dichroism. Calorimetric and fluorescence experiments have shown that Cys76 is responsible for recognition and proton retrieval of D-isomers, while Cys181 is responsible for L-isomer recognition and racemization. This recognition process is further supported by measurements of protein stability followed by chemical denaturation in the presence of the corresponding compound.     

Cloning, sequencing, and expression in Escherichia coli of the D-hydantoinase gene from Pseudomonas putida and distribution of homologous genes in other microorganisms.

Pseudomonas putida DSM 84 produces N-carbamyl-D-amino acids from the corresponding D-5-monosubstituted hydantoins. The gene encoding this D-hydantoinase enzyme was cloned and expressed in Escherichia coli. The nucleotide sequence of the 1.8-kb insert of subclone pGES19 was determined. One open reading frame of 1,104 bp was found and was predicted to encode a polypeptide with a molecular size of 40.5 kDa. Local regions of identity between the predicted amino acid sequence and that of other known amidohydrolases (two other D-hydantoinases, allantionase and dihydroorotase) were found. The D-hydantoinase gene was used as a probe to screen DNA isolated from diverse organisms. Within Pseudomonas strains of rRNA group I, the probe was specific. The probe did not detect D-hydantoinase genes in pseudomonads not in rRNA group I, other bacteria, or plants known to express D-hydantoinase activity.     

Production of D-amino acid using whole cells of recombinant Escherichia coli with separately and coexpressed D-hydantoinase and N-carbamoylase

We developed a fully enzymatic process employing D-hydantoinase and N-carbamoylase for the production of D-amino acid from 5'-monosubstituted hydantoin. For the comparison of the reaction systems using two sequential enzymes, D-hydantoinase of Bacillus stearothermophilus SD1 and N-carbamoyl-D-amino acid amidohydrolase (N-carbamoylase) of Agrobacterium tumefaciens NRRL B11291 were separately expressed in each host cell and coexpressed in the same host cell. A high level and constitutive expression of both enzymes in Escherichia coli in a soluble form was achieved using a promoter derived from B. stearothermophilus SD1. The expression levels of both enzymes ranged from 17% to 23% of the total soluble protein, depending on the expression system. In the case of employing separately expressed enzymes, the product yield of D-hydroxyphenylglycine from D,L-p-hydroxyphenylhydantoin and productivity were 71% and 2.57 mM/g-cell/h in 15 h, respectively. The accumulation of N-carbamoyl-D-hydroxyphenylglycine was significant over the reaction time. On the other hand, use of coexpressed enzymes resulted in 98% product yield of D-hydroxyphenylglycine in 15 h, minimizing the level of intermediates in the reaction mixture. The productivity of coexpressed whole cell reaction was estimated to be 6.47 mM/g-cell/h in 15 h. The coexpressed system was tested for an elevated concentration of D,L-p-hydroxyphenylhydantoin, and efficient production can be achieved.     

Purification and characterization of the hydantoin racemase of Pseudomonas sp. strain NS671 expressed in Escherichia coli.

The hydantoin racemase gene of Pseudomonas sp. strain NS671 had been cloned and expressed in Escherichia coli. Hydantoin racemase was purified from the cell extract of the E. coli strain by phenyl-Sepharose, DEAE-Sephacel, and Sephadex G-200 chromatographies. The purified enzyme had an apparent molecular mass of 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By gel filtration, a molecular mass of about 190 kDa was found, suggesting that the native enzyme is a hexamer. The optimal conditions for hydantoin racemase activity were pH 9.5 and a temperature of 45 degrees C. The enzyme activity was slightly stimulated by the addition of not only Mn2+ or Co2+ but also metal-chelating agents, indicating that the enzyme is not a metalloenzyme. On the other hand, Cu2+ and Zn2+ strongly inhibited the enzyme activity. Kinetic studies showed substrate inhibition, and the Vmax values for D- and L-5-(2-methylthioethyl)hydantoin were 35.2 and 79.0 mumol/min/mg of protein, respectively. The purified enzyme did not racemize 5-isopropylhydantoin, whereas the cells of E. coli expressing the enzyme are capable of racemizing it. After incubation of the purified enzyme with 5-isopropylhydantoin, the enzyme no longer showed 5-(2-methylthioethyl)hydantoin-racemizing activity. However, in the presence of 5-(2-methylthioethyl)hydantoin, the purified enzyme racemized 5-isopropylhydantoin completely, suggesting that 5-(2-methylthioethyl)hydantoin protects the enzyme from inactivation by 5-isopropylhydratoin. Thus, we examined the protective effect of various compounds and found that divalent-sulfur-containing compounds (R-S-R' and R-SH) have this protective effect.     

One-step production of D-p-hydroxyphenylglycine by recombinant Escherichia coli strains

The gene encoding D-hydantoinase from Agrobacterium radiobacter NRRL B11291 was successfully cloned by use of polymerase chain reaction. A positive clone was scored, and its nucleotide sequence was further analyzed. The analysis by deleting various lengths of nucleotides from the amino terminus of the open reading frame revealed the putative regions for promoter and RBS site. By highly expressing both D-hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert DL-hydroxyphenyl hydantoin (DL-HPH) to D-p-hydroxyphenylglycine (D-HPG) with a conversion yield of 97%, accounting for productivity 5 times higher than that obtained by A. radiobacter NRRL B11291. Immobilizing the recombinant cells with kappa-carrageenan could also achieve a conversion of 93%, while A. radiobacter NRRL B11291 attained 20% within the same period of reaction time. These results illustrate the feasibility in employing recombinant E. coli to accomplish one-step conversion of DL-HPH to D-HPG. In the process of improving D-HPG production, D-hydantoinase activity was increased 2.57-fold but carbamoylase activity remained constant, which resulted in only a 30% increase in the reaction rate. It suggests that carbamoylase is the step setting the pace of the reaction. Since the reaction substrate is highly insoluble, achieving sufficient agitation appears to be an important issue in this heterogeneous system. This view is further supported by the study on repeated use of cells, which shows that to reach a conversion of more than 90% free cells can be recycled six times, whereas immobilized cells can be used only twice. In conclusion, the poor reusability of immobilized cells is due to the fouling on the gel surface.     

Molecular cloning and expression of the hyu genes from Microbacterium liquefaciens AJ 3912, responsible for the conversion of 5-substituted hydantoins to alpha-amino acids, in Escherichia coli

A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to alpha-amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl alpha-amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomyces cerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.     

Production of D-p-hydroxyphenylglycine by N-carbamoyl-D-amino acid amidohydrolase-overproducing Escherichia coli strains.

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene from Agrobacterium radiobacter NRRL B11291 has been successfully cloned and expressed in Escherichia coli. Subcloning of the D-carbamoylase gene into different types of vectors and backgrounds of E. coli strains showed that the optimal expression level of D-carbamoylase was achieved in a ColE1-derived plasmid with a 150-fold increase in specific enzyme activity compared to that in a pSC101-derived plasmid. In addition, the recombinant plasmids were very stable in the E. coli strain ATCC11303 but not in JCL1258 tested here. Employing the recombinant E. coli strain DH5alpha/pAH61 for D-p-hydroxyphenylglycine production showed that the cell was capable of transforming N-carbamoyl-D-hydroxylphenylglycine to D-p-hydroxyphenylglycine with a molar conversion yield of 100% and a production rate of 1.9 g/(L h). In comparison with A. radiobacter NRRL B11291, this productivity approximates a 55-fold increase in D-hydroxyphenylglycine production. This result suggests the potential application of recombinant E. coli strains for the transformation reaction.     

Substrate- and stereospecificity,induction and metallo-dependence of a microbial hydantoinase

Summary D, L-5-monosubstituted hydantoins can be used as substrates for a two-step-enzymatic production of optically active aminoacids. The substrate- and stereospecificity of the first enzyme — a hydantoinase -, investigations on its induction and on its dependence upon metallo-ions are described. It is shown, that the activity of this hydantoinase, which is not identical with the well-known enzyme D-hydantoinase, depends on manganese-ions. Of synthetic and natural compounds tested as inductors, D, L-5-indolylmethylhydantoin showed the best effect. The hydantoinase has a wide substrate-specificity. Its stereoselectivity seems to depend on the structure of the side chain in 5-position of the hydantoin.     

Chaperone-assisted overexpression of an active D-carbamoylase from Agrobacterium tumefaciens AM 10.

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene (dcb) from Agrobacterium tumefaciens AM 10 was cloned by polymerase chain reaction in plasmid pET28a and was overexpressed in Escherichia coli JM109 (DE3). However, almost 80% of the enzyme remained trapped in inclusion bodies. To facilitate the expression of the properly folded active enzyme, the chaperones GroEL/ES were coexpressed in plasmid pKY206. This resulted in a 43-fold increase in active enzyme production compared to the wild-type strain. The histidyl-tagged D-carbamoylase was purified by a single step nickel-affinity chromatography to a specific activity of 9.5 U/mg protein.     

The hydantoin transport protein from Microbacterium liquefaciens

The gene hyuP from Microbacterium liquefaciens AJ 3912 with an added His6 tag was cloned into the expression plasmid pTTQ18 in an Escherichia coli host strain. The transformed E. coli showed transport of radioisotope-labeled 5-substituted hydantoins with apparent K(m) values in the micromolar range. This activity exhibited a pH optimum of 6.6 and was inhibited by dinitrophenol, indicating the requirement of energy for the transport system. 5-Indolyl methyl hydantoin and 5-benzyl hydantoin were the preferred substrates, with selectivity for a hydrophobic substituent in position 5 of hydantoin and for the l isomer over the d isomer. Hydantoins with less hydrophobic substituents, cytosine, thiamine, uracil, allantoin, adenine, and guanine, were not effective ligands. The His-tagged hydantoin transport protein was located in the inner membrane fraction, from which it was solubilized and purified and its identity was authenticated.     

微生物乙内酰脲酶及其研究进展

乙内酰脲酶是广泛分布在微生物中的一类可降解乙内酰脲酶类化合物的酶系 ,包括乙内酰脲水解酶、N-氨甲酰氨基酸水解酶及乙内酰脲消旋酶。微生物的乙内酰脲酶在结构与组成、立体选择性、底物专一性、反应条件和作用机制等方面有所不同 ,在各种 L-及 D-型氨基酸的酶法生产中具有良好的应用前景。本文对乙内酰脲酶研究及应用的一般情况作了概述 ,并讨论了有关乙内酰脲酶研究的主要研究进展     

Identification and sequencing of a gene encoding a hydantoin racemase from the native plasmid of Pseudomonas sp. strain NS671.

DNA fragments containing the genes involved in the conversion of 5-substituted hydantoins to their corresponding L-amino acids have been cloned from the 172-kb native plasmid (pHN671) of Pseudomonas sp. strain NS671. The largest recombinant plasmid, designated pHPB14, encoded the ability to convert D-5-substituted hydantoins to the corresponding L-amino acids, whereas the smallest one, designated pHPB12, encoded the ability to convert them to their corresponding N-carbamyl-D-amino acids. Restriction analysis suggested that the inserts of both recombinant plasmids are derived from the identical portion in pHN671 and that the insert of pHPB14, compared with that of pHPB12, has an extra 5.3 kb in length. DNA sequencing revealed that pHPB14 contains two additional complete open reading frames, designated ORF5 and hyuE. Analysis of deletion derivatives of pHPB14 indicated that hyuE is required for the ability to produce L-amino acids from the corresponding D-5-substituted hydantoins, but ORF5 is not. Cells of Escherichia coli transformed with a plasmid containing hyuE were capable of racemizing different 5-substituted hydantoins, indicating that hyuE is a gene encoding a hydantoin racemase.     

A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and L-N-carbamoylase activities as biocatalyst for the production of L-homophenylalanine

A dihydropyrimidinase gene (pydB) was cloned from the moderate thermophilic Brevibacillus agri NCHU1002 and expressed in Escherichia coli. The purified dihydropyrimidinase exhibited strict d-enantioselectivity for D,L-p-hydroxyphenylhydantoin and D,L-5-[2-(methylthio)ethyl]hydantoin, and non-enantiospecificity for D,L-homophenylalanylhydantoin (D,L-HPAH). The hydrolytic activity of PydB was enhanced notably by Mn2+, with a maximal activity at 60 degrees C and pH 8.0. This enzyme was completely thermostable at 50 degrees C for 20 days. A whole cell biocatalyst for the production of L-homophenylalanine (L-HPA) from D,L-HPAH by coexpression of the pydB gene and a thermostable L-N-carbamoylase gene from Bacillus kaustophilus CCRC11223 in E. coli JM109 was developed. The expression levels of dihydropyrimidinase and L-N-carbamoylase in the recombinant E. coli cells were estimated to be about 20% of the respective total soluble proteins. When 1% (w/v) isopropyl-beta-D-thiogalactopyranoside-induced cells were used as biocatalysts, a conversion yield of 49% for L-HPA with more than 99% ee could be reached in 16 h at pH 7.0 from 10mM D,L-HPAH. The cells can be reused for at least eight cycles at a conversion yield of more than 43%. Our results revealed that coexpression of pydB and lnc in E. coli might be a potential biocatalyst for L-HPA production.