Cloning, nucleotide sequence and expression of a ... - Springer Link

Appl Microbiol Biotechnol (2001) 55:750–757 DOI 10.1007/s002530000574

O R I G I N A L PA P E R

Anja Wiese · Burkhard Wilms · Christoph Syldatk Ralf Mattes · Josef Altenbuchner

Cloning, nucleotide sequence and expression of a hydantoinase and carbamoylase gene from Arthrobacter aurescens DSM 3745 in Escherichia coli and comparison with the corresponding genes from Arthrobacter aurescens DSM 3747 Received: 27 July 2000 / Received revision: 13 November 2000 / Accepted: 17 November 2000 / Published online: 5 May 2001 © Springer-Verlag 2001

Abstract The genes encoding hydantoinases (hyuH1) and carbamoylases (hyuC1) from Arthrobacter aurescens DSM 3745 and Arthrobacter aurescens DSM 3747 (hyuH2, hyuC2) were cloned in Escherichia coli and the nucleotide sequences determined. The hydantoinase genes comprised 1,377 base pairs and the carbamoylase genes 1,239 base pairs each. Both hydantoinases, as well as both carbamoylases, showed a high degree of nucleotide and amino acid sequence identity (96–98%). The hyuH and hyuC genes were expressed in E. coli under the control of the rhamnose promoter and the different specific activities obtained in E. coli crude extracts were compared to those produced by the original hosts. For purification the hyuH2 gene was expressed as a maltosebinding protein (MalE) and as an intein–chitin binding domain (CBD) fusion in E. coli. The expression of malE-hyuH2 resulted in the production of more soluble and active protein. With respect to temperature stability, optimal pH and optimal temperature, substrate and stereospecificity, the purified fusion enzyme exhibited properties similar to those of the wild-type enzyme.

Introduction The genus Arthrobacter belongs to the group of actinomycetes which have an extensive secondary metabolism including the ability to transform steroids and to produce antibiotics, fungicides, herbicides, immunosupressives, pigments, phytohormones, oligo- and polysaccharides and amino acids. The particular A. aurescens strains A. Wiese · B. Wilms · R. Mattes · J. Altenbuchner (✉) Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany e-mail: [email protected] Tel.: +49-711-6857591, Fax: +49-711-6856973 C. Syldatk Institut für Bioverfahrenstechnik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany

DSM 3745 and DSM 3747 are capable of stereospecifically converting 5-monosubstituted hydantoins to α-amino acids (Gross et al. 1987) and may be useful for production of enantiomeric pure amino acids for use as pharmaceuticals, agrochemicals and food supplements. The bacterial conversion of racemic 5-monosubstituted hydantoins needs three steps, an enantioselective hydantoin ring opening by hydantoinase, the enantiospecific hydrolysis of the intermediate N-carbamoyl amino acid to the α-amino acid by carbamoylase and the racemication of the 5-monosubstituted hydantoins by the hydantoin racemase. Together, the three steps allow a complete conversion of racemic hydantoins to optically pure amino acids (reviewed by Ogawa and Shimizu 1997; Syldatk et al. 1999). The hydantoinase from A. aurescens DSM 3745 was purified by May et al. (1998a) to homogeneity and is well characterized. The enzyme was shown to be a Zn2+metalloenzyme (May et al. 1998c, d) which is L-selective for the cleavage of D,L-5-(3′-indolylmethyl)-hydantoin (IMH). According to its amino acid sequence (SwissProt accession number P81006) the enzyme belongs to the superfamily of amidohydrolase (May et al. 1998b). The second well-characterized enzyme is the carbamoylase from A. aurescens DSM 3747. The gene has been cloned, sequenced, expressed in Escherichia coli, and the protein purified and characterized (Wilms et al. 1999). In order to improve enzyme production and enzyme properties, it is most helpful to have the corresponding genes cloned. In this report, we describe the cloning and DNA sequencing of the hydantoinase as well as the carbamoylase gene from A. aurescens DSM 3745. The genes and the corresponding enzymes were compared with those from A. aurescens DSM 3747 regarding their nucleotide and amino acid sequences as well as the enzyme activities obtained by homologous and heterologous expression. Furthermore the heterologous expression of the hydantoinase gene from A. aurescens DSM

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3747 and purification of the enzyme was improved by protein fusion with the E. coli maltose binding protein MalE.

Materials and methods Bacterial strains, plasmids and growth conditions Strains and plasmids used or constructed in this study are shown in Table 1. The A. aurescens strains were cultivated at 30°C in yeast extract medium K1 (Gross et al. 1987) containing 0.3 g l–1 3-N-methyl-5-(3′-indolylmethyl)-hydantoin as an inducer. E. coli strains were grown in 2×YT medium (Sambrook et al. 1989) supplemented with appropriate antibiotics (ampicillin 100 µg ml–1, kanamycin 50 µg ml–1) at 37°C. Recombinant DNA techniques All of the recombinant DNA techniques used were standard methods (Sambrook et al. 1989). Restriction enzymes and ligase were purchased from either Roche Molecular Biochemicals (Mannheim, Germany) or New England BioLabs (Frankfurt, Germany) and used as recommended by the manufacturer. DNA restriction fragments used in cloning were eluted from the agarose using the gel extraction kit JETSORB (Genomed, Bad Oeynhausen, Germany. Expression of hyuH, hyuC, malE-hyuH2 and hyuH2-CBD For HyuH production in E. coli the hyuH genes were amplified by using the primers S956 (5′-AGA ACA TAT GTT TGA CGT AAT AGT TAA GAA-3′) and S957 (5′-AAA AGG ATC CTC ACT TCG ACG CCT CGTA-3′), chromosomal DNA as template and the Taq DNA-polymerase (Biomaster, Cologne, Germany). For amplification of the carbamoylase genes the primers S1066 (5′AGA ACA TAT GAC CCT GCA GAA AGC G-3′) and S1067 (5′AAA AGG ATC CTT ACC GTT CAA GTG CCT T-3′) were used. The PCR fragments were inserted between the NdeI and BamHI sites of the expression vector pJOE2702 (Volff et al.

1996), resulting in the plasmids pAW191 (hyuH1), pAW92 (hyuH2), pAW173 (hyuC1) and pAW178-2 (hyuC2; Wilms et al. 1998). The gene hyuH2 was also expressed in E. coli as a maltose binding (MalE) and an intein–chitin binding domain (CBD) fusion protein, respectively. A new vector with malE and an L-rhamnose inducible rhaB promoter was constructed. The malE sequence without the signal sequence for export was amplified from pMALc2 vector (New England Biolabs) using the primers 5′-AAA AGG ATC CCC TTC CCT CGA TCC CGA GGT T-3′ and 5′-AAA ACA TAT GAA AAC TGA AGA AGG TAA ACT G-3′. The resulting fragment was cut with NdeI and BamHI and inserted into pJOE2702 to obtain pJOE2955. The oligonucleotides used in the PCR reaction to amplify the hyuH2 gene were S957 (see above) and S1136 (5′-AGA AGG ATC CAT GTT TGA CGT AAT AGT TAA GA-3′) and the fragment was inserted into the BamHI site of pJOE2955. The resulting plasmid pAW211 was transformed in E. coli JM109. The construction of the expression vector with CBD was based on pTYB2 (IMPACT, New England Biolabs). The 1.6 kb NdeI/PstI fragment of pTYB2, containing intein and CBD, was inserted between the corresponding sites in pJOE2702 yielding pAW280. The gene hyuH2 was PCR amplified with the primers S956 (see above) and S2253 (5′-AAA ACC CGG GCT TCG ACG CCT CGT AGT G-3′), the fragment cut with NdeI and SmaI and inserted into pAW280 which was cut with the same enzymes. In this expression vector pAW302 the intein–CBD encoding gene was fused with the C-terminal end of the hydantoinase gene. Strains of E. coli JM109 carrying the various expression plasmids were grown in 2×YT. The cells were shifted to a growth temperature of 30°C at an OD600=0.3 to reduce the formation of inclusion bodies and L-rhamnose was added a final concentration of 0.2% for induction of the rhamnose promoter. The cells were further incubated for several hours at 30°C before crude extracts were prepared. Enzyme assay Hydantoinase activity was determined by measuring formation of N-carbamoyl tryptophan (N-CTrp) from D,L-IMH by HPLC (Thermoseperation Products, Darmstadt, Germany). One unit of activity is defined as 1.0 µmol N-CTrp per min. Cells were suspended in 0.2 M Tris-HCl, pH 7.2 and 1 mM MnCl2. E. coli cells were sonicated (ultrasonic processor W-385, Heatsystems Ultrasonic, Farmingdale, USA) twice for 30 s and centrifuged at

Table 1 Bacterial strains and plasmids used Strains A. aurescens DSM 3745 A. aurescens DSM 3747 E. coli JM109 Plasmids pAW16 pAW92 pAW173 pAW178-2 pAW191 pAW211 pAW280 pAW302 pIC20H pJOE2702 pJOE2955 pMAL-c2 pTYB2

Relevant characteristics

recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ∆(lac-proAB) F'[traD36 proAB+ lacIq lacZ∆M15] Genomic library plasmid containing the hyu genes of Arthrobacter aurescens DSM 3747; KmR hyuH1, amplified by PCR, inserted in pJOE2702 hyuC1, amplified by PCR, inserted in pJOE2702 hyuC2, amplified by PCR, inserted in pJOE2702 hyuH2, amplified by PCR, inserted in pJOE2702 hyuH2, amplified by PCR, inserted in pJOE2955 intein and cbd of pTYB2 inserted between the NdeI and PstI site of pJOE2702 hyuH2, amplified by PCR, inserted in pAW280 Cloning vector, Amp Expression vector with rha BAD promoter PCR amplified mal E inserted between the NdeI and BamHI site of pJOE2702 MalE fusion vector Intein-chitin binding domain fusion vector

Reference Gross et al. 1987 Gross et al. 1987 Yanish-Perron et al. 1985

Wiese et al. 2000 This study This study This study This study This study This study This study Marsh et al. 1984 Volff et al. 1996 This study New England Biolabs New England Biolabs

752 15,000 g for 10 min, whereas A. aurescens cells were vortexed with 10 µl toluene for 10 s. Assays were performed by adding 100 µl enzyme solution to 800 µl 0.2 mM D,L-IMH dissolved in 0.1 M Tris-HCl, pH 8.5 at 37°C. After 10 min the reaction was stopped by addition of 400 µl 14% (w/v) trichloroacetic acid and analyzed by HPLC. If more than 10% of the substrate was converted, the assay was repeated with a diluted enzyme solution. Protein concentrations were determined by the method of Bradford (1976), with the Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, Munich, Germany). Standard curves were generated with bovine serum albumin. Crude extracts were analyzed on a 10% SDS-PAGE (Laemmli 1970). Purification of MalE-HyuH2 The purification of the MalE-HyuH2 fusion protein was done by affinity chromatography on amylose resin following the protocol of the manufacturer of the resin (New England Biolabs). JM109 pAW211 was grown in 300 ml 2×YT plus ampicillin, induced with L-rhamnose for 4 h at 30°C, harvested by centrifugation and washed in amylose resin puffer (AR buffer, 20 mM Tris/HCl, 20 mM NaCl, 1 mM MnCl2, 0.2 mM NaN3, pH 7.4). The cells were resuspended in 3 ml AR buffer and disrupted by using a French Press (800–1,000 psi). Cell debris was removed by centrifugation (15,000 g, 10 min, 4°C) and the supernatant diluted with AR buffer to a protein concentration of 2.5 mg ml–1. Amylose resin (15 ml) was filled in a column (1×20 cm) and equilibrated with 100 ml AR buffer. The protein solution (18 ml) was loaded on to the column at a flow rate of 1 ml min–1 using a peristaltic pump (P1, Pharmacia, Freiburg, Germany). The resin was washed with 150 ml AR buffer and the fusion protein eluted with 15 ml ARbuffer containing 10 mM maltose. Fractions with hydantoinase activity were combined and directly used for further characterization. Characterization of MalE-HyuH2 The pH profile of the purified enzyme was measured in the pH range 7.5–11.5. The substrate was dissolved in Tris/HCl buffer (pH 7.5–9.0) and in glycine/NaOH buffer (pH 8.5–11.5). Enzyme activity was determined as described above. The reaction temperature optimum was analyzed at temperatures between 25°C and 65°C using the standard enzyme assay. The stability of the fusion protein was measured after preincubation at temperatures between 25°C and 60°C for 30 min in the presence of 0.2 M Tris-HCl, pH 7.2 and 1 mM MnCl2.

Fig. 1a–c Growth curve of Arthrobacter aurescens DSM 3745 (open circles) and A. aurescens DSM 3747 (solid circles) in yeast extract medium K1 with 3-N-CH3-IMH at 30°C (a). Specific activities of hydantoinase (b) and carbamoylase (c) in A. aurescens DSM 3745 (open bars) and DSM 3747 (solid bars) during the cultivation with 3-N-CH3-IMH. Activities were determined in toluene-treated cells using D,LIMH and N-CTrp, respectively, as substrates

Results Hydantoinase and carbamoylase activity of A. aurescens DSM 3745 and DSM 3747 Both A. aurescens DSM 3745 and DSM 3747 were isolated as hydantoin hydrolyzing microorganisms (Gross et al. 1987). The two strains differed in their cytochrome C oxidase activity and in their streptomycin and gentamycin resistance (Höke et al. 1988). Moreover the genetic variation between the hydantoinase gene clusters of the two Arthrobacter strains was proven by restriction fragment length polymorphism (data not shown). Similar generation rates of both Arthrobacter strains were monitored in K1-medium which contains 3-N-CH3-IMH, the inducer of hydantoinase and carbamoylase activity (Fig. 1a). Hydantoinase and carbamylase activities in crude extracts of both induced strains were compared. A. aurescens DSM 3745 exhibited a 2- to 5-fold higher hydantoinase and carbamoylase activity, respectively, in comparison to A. aurescens DSM 3747 (Fig. 1b, c). Comparison of the hydantoinase and carbamoylase gene sequences The hydantoinase gene hyuH2 and carbamoylase gene hyuC2 of A. aurescens DSM 3747 were obtained from pAW16, a plasmid from a genomic library in the λRESIII vector (Altenbuchner 1993) which was screened with a labeled oligonucleotide deduced from the N-terminal amino acid sequence of the A. aurescens DSM 3735 hydantoinase. Sequencing of a 7 kb fragment revealed the presence of a hydantoinase gene, a carbamoylase gene, a racemase gene and a putative hydantoin uptake system (Genbank accession no. AF146701). Oligonucleotides deduced from the N- and C-terminal regions of A. aurescens DSM 3747 hydantoinase and carbamoylase gene were designed to amplify the genes hyuH1 and hyuC1 from A. aurescens DSM 3745. The genes were cloned in

753 Fig. 2 Induction kinetics of the carbamoylase genes in Escherichia coli JM109 pAW178-2 (HyuC2; solid bars) and pAW173 (HyuC1; open bars), after induction with 0.2% rhamnose (top). The specific activities were determined with N-CTrp as substrate. SDSPAGE analysis of cell extracts of JM109 pJOE2702 (vector, lane 2, 3), JM109 pAW173 (lane 4, 5) and JM109 pAW178-2 (lane 6, 7), grown for 8 h without (lanes 2, 4, 6) or with inducer (lanes 3, 5, 7) (below). The crude extract was separated by centrifugation in soluble (left gel) and insoluble (right gel) fraction. The carbamoylase proteins are marked by triangles. Lane 1: molecular mass standard (kDa)

the E. coli vector pIC20H (Marsh et al. 1984) and were subsequently sequenced. The sequences of the two hydantoinase genes from the A. aurescens strains differed in at least 46 nucleotides (further differences might be in the regions were the oligonucleotides bound). This led to an amino acid exchange at 8 positions and gives an amino acid sequence identity of 98.2%. May et al. (1998b) already showed that HyuH1 belongs to the amidohydrolase superfamily which was discovered by Holm and Sanders (1997). The differences in the nucleotide sequences of the carbamoylase genes isolated from the two A. aurescens strains were similar to the hydantoinase genes. Altogether, 52 nucleotides were exchanged, resulting in 13 changes of amino acids in the deduced polypeptides or 97% identity of the two proteins. Expression of the hyuC and hyuH genes in E. coli The hyuC2 gene was amplified with the oligonuclotides S1066 and S1067 adding a NdeI restriction site at ATG start codon and a BamHI site just after the stop codon of hyuC2. This allowed the precise fusion of the gene to the ribosomal binding site in the rhamnose inducible vector pJOE2702 as described by Wilms et al. (1999). The hyuC1 gene was amplified using the same primers and again inserted into the vector pJOE2702. The two plasmids pAW178-2 (hyuC2) and pAW173 were brought into

E. coli JM109, the cells grown in 2×YT medium and induced with 0.2% rhamnose at 30°C growth temperature. Expression of hyuC2 in E. coli gives high amounts of active carbamoylase even so a considerable part of the enzyme is produced as inactive inclusion bodies. Remarkably, the specific carbamoylase activity increased steadily within 8–10 h of induction; even so, the cells had already reached the stationary growth phase as shown by Wilms et al. (1999) and a specific enzyme activity of about 1.5 units (mg protein)–1 was obtained in the crude extract using N-CTrp as substrate. This corresponds to a 50- to 100-fold increase of enzyme activity compared to the A. aurescens strains. With JM109 pAW173 containing the carbamoylase gene from A. aurescens DSM 3745 significant lower enzyme activities were obtained. The enzyme activity increased within about 5 h induction with rhamnose to a maximum value of about 0.65 units (mg protein)–1 and after that decreased again. The amount of protein produced by the recombinant cells was about the same (Fig. 2) but the crude extract from JM109 pAW178-2 contained a slightly higher amount of soluble and lower amount of insoluble carbamoylase, respectively, as determined by densitometry of the SDS-gel (data not shown). This alone does not explain the twofold difference seen in the enzyme activity and so one has to conclude that the carbamoylase of A. aurescens DSM 3745 has a lower specific activity towards the substrate N-CTrp compared with HyuC2. The hyuH1 and hyuH2 genes were amplified by PCR as well by using primers S956 and S957, which intro-

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tein was already produced as inclusion bodies. The proportion of soluble protein seemed to be higher in JM109 pAW191 (HyuH1) than in JM109 pAW92 (HyuH2). After an induction of 8 h, JM109 pAW92 and JM109 pAW191 exhibited specific hydantoinase activities of 0.35 U (mg protein)–1 and 0.75 U (mg protein)–1, respectively (Fig. 3). In comparison, in crude extracts of A. aurescens maximum hydantoinase activities of 0.1 U (mg protein)–1 for HyuH2 and 0.17 U (mg protein)–1 for HyuH1, respectively, were obtained within 24 h of cultivation (Fig. 1). The specific activities in the E. coli crude extracts turned out to be 3- to 4-fold higher than in those obtained with the A. aurescens strains DSM 3745 and DSM 3747. Expression studies of hydantoinase fused with maltose binding protein and intein–chitin binding domain

Fig. 3 Hydantoinase activity in crude extracts of E. coli pAW92 (solid bars) and JM109 pAW191 (open bars) (top). The cells were grown in 2×YT at 30°C and induced with 0.2% rhamnose. SDSPAGE analysis of crude extracts from E. coli JM109 pJOE2702 (C, vector control), JM109 pAW92 and JM109 pAW191, uninduced (–) or induced up to 10 h (below). The cell crude extract was separated by centrifugation in soluble (S) and insoluble (P) fractions. Lane M: Molecular mass standard (kDa)

duced an NdeI site at the ATG start codon and a BamHI site just behind the stop codon. The fragments were inserted in pJOE2702 under the control of a rhamnose-inducible E. coli promoter as described for the carbamoylase genes. The resulting plasmids were designated as pAW191 (hyuH1) and pAW92 (hyuH2), respectively. Crude extracts from transformed E. coli JM109 cells carrying the plasmids pAW92 and pAW191 were analyzed by SDS-PAGE, confirming the formation of a polypeptide with the correct molecular mass of about 50 kDa (Fig. 3). After an induction period of 2 h most of the pro-

For one-step affinity purifications of recombinant proteins, several expression and purification systems are commercially available. The most common method using proteins fused with six histidine residues was not appropriate for the purification of the hydantoinase since the protein was inactivated by the eluent imidazole, presumably through removal of the Zn2+ ion. The alternative elution of the hydantoinase at low pH inactivated the enzyme as well (data not shown). The fusion to maltose binding protein MalE allows purification of the fusion protein by affinity chromatography on composite amylose–agarose beads. In addition, it was reported that the fusion to MalE enhances the solubility of some proteins (Georgiou and Valax 1996). In order to facilitate the soluble expression of hyuH, the hydantoinase gene (hyuH2) from A. aurescens DSM 3747 was fused with the malE gene. In analogy to the expression vectors pMal from New England Biolabs the vector pJOE2955 was constructed, which enables the expression of the gene fusions under the control of the rhamnose promoter. The malE gene with a recognition site for protease factor Xa at the C-terminal end but without the sequence encoding the signal protein for export was amplified by PCR from plasmid pMAL-c2 using primers which introduced an NdeI site at the start codon and a BamHI site behind the factor Xa cleavage site. The fragment was inserted into pJOE2702 between the NdeI and BamHI sites of the vector (pJOE2955). The hyuH2 gene was amplified with oligonucleotides, introducing BamHI sites just upstream the start codon and downstream the stop codon. The fragment was inserted into the BamHI site of pJOE2955 (yielding plasmid pAW211) which resulted in a fusion of the two genes. Alternatively, an expression vector pAW302 with rhamnose promoter was constructed including a hyuH2 fusion with the intein and chitin binding domain based on the IMPACT system from New England Biolabs. Thus the intein–chitin binding domain was fused to the C-terminal end of the hydantoinase. The chitin binding domain allows a one-step affinity purification of the fusion protein on chitin containing beads. Af-

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Fig. 4 Kinetics of induction of hydantoinase activity in E. coli JM109 with plasmids pAW92 (open bars), pAW211 (shaded bars) and pAW302 (solid bars) (top). Activity was determined in crude extracts by standard hydantoinase enzyme assays. E. coli cells were cultivated in 2×YT at 30°C and induced with 0.2% rhamnose. SDS-PAGE of soluble (S) and insoluble fractions (P) of JM109 pJOE2702 (vector control, lane 1, 2), JM109 pAW92 (HyuH2, lanes 3–6), JM109 pAW211 (MalE-HyuH2, lanes 7–10) and JM109 pAW302 (HyuH2-intein-CBD, lanes 11, 12, 13, 14) (below). Crude extracts were prepared from cells induced for 4 h with rhamnose from cells grown without inducer. Lanes 1, 3, 5, 7, 9, 11, 13 represent the crude extract from uninduced, lanes 2, 4, 6, 8, 10, 12, 14 from rhamnose-induced cells. Lane M: molecular mass standard (kDa)

ter binding, the fusion is then induced to undergo inteinmediated self-cleavage releasing the hydantoinase. The hydantoinase activities of JM109 harboring either pAW92, pAW211 or pAW302 were determined after induction with rhamnose at 30°C in 2×YT supplemented with ampicillin. The MalE–HyuH fusion exhibited the highest enzyme activity of 0.75 U (mg protein)–1 (Fig. 4). The analysis of the soluble and insoluble fractions by SDS-PAGE was in accordance with the determined enzyme activities (Fig. 4). The HyuH–intein–CBD fusion turned out to be even less soluble then the unfused HyuH protein and was not investigated further.

Fig. 5a–c Effect of pH and temperature on hydantoinase activity of purified MalE-HyuH2. a Temperature optimum: the standard enzyme assay was performed at different temperatures. b Temperature stability: the residual activity was measured after incubating for 30 min at different temperatures. c pH optimum: Tris/HCl buffer was used for pH 7.5–9.0 (squares), and glycine/NaOH buffer for pH 8.5–11.5 (triangles)

Enzymatic properties of the fusion protein MalE-HyuH2 The purification of the fusion protein MalE-HyuH2 was based on the protein purification protocol developed by New England Biolabs, and is described in the Materials and methods section and in Pietzsch et al. (2000). The proteolytic cleavage with factor Xa was ineffective, which has also been shown for other MalE fusions (Grob and Guiney 1996). Thus the uncleaved MalE-HyuH2 was enzymatically characterized. The native fusion protein was identified like the wild-type enzyme as a homotetramer by size exclusion separation on a superose 12 column (O. May, personal communication). The effect of temperature on the enzyme activity was examined, and for MalE-HyuH2 the optimal reaction temperature was found to be about 45°C (Fig. 5a). The thermal stability was evaluated by measuring the residual enzyme activity after incubation for 30 min at different temperatures. A denaturation of the fusion protein occurred at temperatures higher than 45°C (Fig. 5b). The dependence of enzyme activity on pH was also investigated. The activity of MalE-HyuH2 was at a maximum at pH 9.5 (Fig. 5c).

756 Table 2 Substrate and stereospecificity of the fusion protein MalE-HyuH Substrate

Concentration (mM)

Relative activity (%)

D-IMH L-IMH D/L-IMH D-MTEH L-MTEH D/L-MTEH

2 2 2 20 20 20

2 100a 71 13 5 9

a The specific activity of the purified MalE-HyuH2 was 12.8 U (mg protein)–1 with D,L-IMH as substrate and tested under standard conditions. The protein was more than 95% pure as charged from SDS-PAGE

These results were very similar to those observed with the enzyme purified from A. aurescens DSM 3747. Here the temperature optimum was about 50°C, thermal stability about 46°C and pH optimum between pH 8.8 and 9.25 (Syldatk et al. 1992). For further characterization of the fusion protein the substrate specificity for IMH and MTEH was investigated. The recombinant fusion protein had a preference for the aromatic substrate as described for the wild-type enzyme (Table 2). The cleavage was found to be L-selective for IMH and D-selective for MTEH. These findings correlate with the substrate and stereospecificity of the wild-type enzyme from A. aurescens DSM 3745 (May et al. 1998a).

Discussion Arthrobacter aurescens DSM 3745 and DSM 3747 are able to convert 5-monosubstituted hydantions enantioselectively into α-amino acids by employing hydantoinase, carbamoylase and racemase. The hydantoinases and carbamoylases of the closely related strains were compared by cloning, sequencing and expression of the genes in E. coli. The two hydantoinases differ in eight amino acids. The enzymes belong to the family of amidohydrolases as already shown for the hydantoinase of A. aurescens DSM 3745 (May et al. 1998b). Members of this family are D- and L-hydantoinases, dihydropyriminidases, dihydroorotases, allantoinases, adenine, AMP and cytosine deaminases, ureases and some other amidohydrolases. The enzymes share the same active-site architecture consisting of a (βα)8 barrel with a conserved amino acid sequence motif for binding of divalent cations (except for three examples involved in the neuronal development of animals) which are essential for the metal-assisted hydrolysis of the amide bond. These highly conserved amino acids, i.e., four histidines and one aspartic acid residue, are also present in the Zn2+ binding hydantoinase from A. aurescens DSM 3745 and can also be found in the sequence of the A. aurescens DSM 3747 hydantoinase. The exchange of the eight amino acids occurred in regions of the enzymes which are not highly conserved within hydantoinases.

The two carbamoylases differ in 13 amino acids. In contrast to the hydantoinases there are no similarities between D- and L-carbamoylases. D-carbamoylases share significant similarities with amidases. Similarities of the two L-carbamoylases were only found to an L-carbamoylase of Pseudomonas sp. (Watabe et al. 1992) and two putative L-carbamoylases of E. coli and Haemophilus influenzae. The similarities were in the order of 30–40% identity in amino acid sequences (Wilms et al. 1999). The differences between the two L-carbamoylases were in regions which are not conserved in this group of enzymes. Hydantoinase and carbamoylase activity has to be induced in A. aurescens by 3-N-CH3-IMH. Reproducibly, A. aurescens DSM 3747 showed about half the hydantoinase and carbamoylase activities of A. aurescens DSM 3745. When the carbamoylase genes were expressed in E. coli, about the same amount of protein was produced. On the other hand, carbamoylase activity obtained from heterologous expression in E. coli was significantly lower with the gene from DSM 3745. In contrast, by expression of the hydantoinase gene of A. aurescens DSM 3747 in E. coli only about half of the enzyme activity was obtained in comparison with the A. aurescens DSM 3745 hydantoinase gene. For the hydantoinases, this seems not to be due to a lower specific activity of the A. aurescens DSM 3747 hydantoinase but to a lower amount of correctly folded protein. Indeed, by fusion of the A. aurescens DSM 3747 hydantoinase gene to the malE, gene the amount of soluble and active enzyme could be considerably increased and the specific activity of the purified MalE-HyuH protein towards D,L-IMH was about the same as that reported for the hydantoinase purified from A. aurescens DSM 3745. In summary, the lower hydantoinase and carbamoylase activity seen in A. aurescens DSM 3747 compared with A. aurescens DSM 3745 seems to be mainly due to a lower expression of the genes and not due to lower specific activities of the enzymes. The bottleneck in using A. aurescens as a whole-cell biocatalyst in producing α-amino acids from hydantoins is clearly the very low carbamoylase activity observed in the two strains. By heterologous expression of the genes in E. coli, the carbamoylase activity was increased between 50- to 100fold, leading to a higher specific activity towards N-CTrp (1.5 units mg–1 for HyuC2). It even turned out that the gene giving lower activity in A. aurescens performed better in the heterologous expression. In the case of the heterologous expression of the hydantoinases, the specific enzyme activities were only increased 2- to 5-fold in comparison with A. aurescens, giving a maximum specific activity towards D,L-IMH of 0.75 units (mg protein)–1 in the crude cell extract. Here the A. aurescens DSM 3745 hydantoinase gene gave the better results. This means that naturally occurring small differences in amino acid sequences may have dramatic effects in the heterologous expression of a gene which are not predictable. The data reported in this study are therefore an important prerequisite for constructing a recombinant

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E. coli whole-cell biocatalyst for the production of αamino acids from racemic hydantoins. Acknowledgements We thank the Deutsche Forschungsgemeinschaft for financial support and the Institut für Bioverfahrenstechnik for kindly providing the HPLC system and hydantoin derivatives. Oliver May is gratefully acknowledged for helpful comments and Kerstin Neumann for technical assistance. We declare that the experiments presented in this paper were done in compliance with the current laws in Germany.

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