Springer 2006
World Journal of Microbiology & Biotechnology (2006) 22:675–680 DOI 10.1007/s11274-005-9088-y
Purification, enzymatic properties of a recombinant D-hydantoinase and its dissociation by zinc ion Ya-Wei Shi, Li-Xi Niu, Xia Feng and Jing-Ming Yuan* Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, 92 Wucheng Road, 030006, Taiyuan, P. R. China *Author for correspondence: Tel.: +86-351-7018268, E-mail:
[email protected] Received 15 August 2005; accepted 31 October 2005
Keywords:
D-hydantoinase,
dissociation, properties, purification, zinc ion
Summary A D-hydantoinase was expressed in the soluble form by a recombinant E. coli strain, pE-HDT/E. coli BL21 in LB medium. The enzymatic activity of cultured cells reached 5.2–6.5 IU/ml culture at a cell turbidity of 10 at 600 nm. The expressed enzyme was efficiently purified by three steps, ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic interaction chromatography and Sephacryl S-200 size-exclusion chromatography. With the above purification process, the enzyme was purified to more than 95% purity as estimated by SDS-PAGE. The overall recovery of enzymatic activity was 54.4% and the specific activity for substrate DL-hydantoin achieved 48 U/mg. The purified enzyme appeared as a dimer with a molecular mass of 103 kDa, as measured by size-exclusion chromatography. The enzyme was stable from pH 6 to 12 with an optimum pH at 9.5 The optimum temperature of the enzyme was 45 C and it activity was rapidly lost over 55 C. Divalent metal ions, including Co2+, Mn2+ and Ni 2+ ions obviously enhanced the enzymatic activity, while Zn2+ ion had a slight inhibitory effect. In addition, the dissociation of purified enzyme into its subunits occurred in the presence of 1 mM Zn2+ ion. The effect of different metal ions on the D-hydantoinase activation/attenuation was discussed.
Introduction Hydantoinase (EC 3.5.2.2) hydrolyses its substrate 5¢-monosubstituted hydantoin to N-carbamoyl-amino acid and in turn, the resultant product can be chemically or enzymatically converted into the corresponding optically active amino acid (Syldatk et al. 1999). According to substrate chirality, hydantoinase can be classified into three types, specific for L-, D- and DL-configuration, which widely exist in animals, plants and microorganisms (Lapointe et al. 1994). However, a number of bacterial D-hydantoinases with different stereo-selectivity and substrate specificity have been used in the industrial bioconversion of optically active D-amino acids (Takahashi et al. 1979; Olivieri et al. 1981). D-Amino acids as unnatural chiral products are important intermediates in the synthesis of various products, such as b-lactam semisynthetic antibiotics, antiviral agents, artificial sweeteners, pesticides, peptide hormones, and pyrethroids (Ogawa & Shimizu 1999). The functional molecule of D-hydantoinase is generally a homologous dimer or a tetramer, which is oligomerized from a subunit with molecular mass of 50–60 kDa. Due to the importance of D-hydantoinase in the industrial bioconversion of amino acids, its biochemical properties,
reaction mechanism and protein conformation have been extensively investigated (Xu et al. 2003; Radha Kishan et al. 2005). Although D-hydantoinases from divergent sources have many common features, certain subtle differences including substrate specificity, optimal pH and temperature, activator and inhibitor etc. are found. Thus the difference in the apparent enzyme activity may be determined by protein conformation. The active site of D-hydantoinase usually contains four histidines, one aspartate and one lysine. Among those amino acid residues, histidines always coordinate with Zn2+ ion to facilitate substrate hydrolysis (Xu et al. 2003). However, the enzyme reported in this study, unlike most D-hydantoinases, can be dissociated into its subunits by Zn2+ ion, resulting in the activity attenuation. The phenomenon seems similar to the observed role of the dinuclear metal center in D-aminoacylase (Liaw et al. 2003). The enzyme molecule may have a TIM barrel metal-dependent structure with a and b-subsites as in D-aminoacylase. During the coordination of the enzyme and substrate, addition of extrinsic Co2+, Ni2+, Mn2+ ions can hold the b-subsite to activate the enzyme, while the additional Zn2+ion tightly binds to the a-subunit to attenuate the enzyme activity. Because zinc ion is one of the most important metal ions in living organism, the
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function of zinc ion in hydantoinase is worth further investigation.
One D-hydantoinase unit is defined as the amount of the enzyme produced 1 lmol of N-carbamoylglycine per min under the above assay condition.
Materials and methods
Protein concentration
Materials
The protein concentration was determined by the Bradford assay, using bovine serum albumin as the standard.
The strain Pseudomonas putida YZ-26 was stored in this laboratory at )70 C. The vector pET-3a was purchased from Invitrogen Co. Phenyl-Sepharose, Sephacryl S-200 and the pre-packed Superose 12(10/30) column were products of Pharmacia. DL-Hydantoin, dihydrouracil, 5¢-phenylhydantoin, uracil and succinimide were purchased from Sigma-Aldrich Fine Chemicals. DL-p-Hydroxyphenyl-hydantoin was synthesized according to the method reported in the literature (Ohashi et al. 1981). Protein marker and restriction enzymes were from New-England BioLabs Co. Other chemicals used were analytical grade. Cloning and expression of the D-hydantoinase gene An ORF corresponding to D-hydantoinase was amplified by PCR using primers (FW: 5¢-GG GGT ACC C ATG TCC CTG TTG ATC CG-3¢ and RV: 5¢-TTA GGA TCC TCA GCG CTG AAC TGG-3¢) with genomic DNA from Pseudomonas putida strain YZ-26 as the template. The resultant PCR product digested by restriction enzyme NdeI and BamHI was inserted into vector pET3a, yielding a recombinant plasmid pE-HDT. The ORF encoding 1440 bp was verified by DNA sequence analysis (TaKaRa, Dalian, China) and has been deposited in GenBank (Accession No. AY 387829). The construct pE-HDT was transformed into E. coli BL21. The engineered strain pE-HDT/E. coli BL21 was grown overnight in Luria–Bertani medium (LB) supplemented with ampicillin (100 lg/ml). Then 2 ml of pre-cultured cells were inoculated into 200 ml LB medium and incubated at 37 C for 10–12 h in an orbital shaker at 200 rev/min. Cells were then harvested by centrifugation at 6000 rev/min at 4 C for 25 min. Enzyme activity assay D-Hydantoinase
activity was measured by a colorimetric method (Takahashi et al. 1978). To start the reaction, an aliquot of the enzyme solution was added into the reaction mixture in a total volume of 1.5 ml, contained 100 mM DL-hydantoin, 50 mM Tris–HCl buffer, pH 8.0. After being incubated at 37 C for 30 min, the reaction mixture was stopped by the addition of 0.25 ml trichloroacetic acid (10% w/v) and 0.25 ml dimethylaminobenzaldehyde solution (10% w/v in 6 M HCl), and then diluted with distilled water to a volume of 3 ml. After centrifugation, the concentration of N-carbamoylglycine in the supernatant was measured at 430 nm and calculated from a standard calibration plot.
Purification of the D-hydantoinase The purification procedure was conducted at 4 C with a LKB chromatographic system, and 50 mM Tris–HCl column buffer, pH 8.0 was used in all steps. Step 1: Preparation of crude extract. The cell pellet from 200 ml E. coli cell culture, harvested by centrifugation at 6000 rev/min, 4 C for 25 min, was washed twice and suspended in chilled column buffer to reach protein concentration at ca. 30 mg/ml. The suspended solution was disrupted with sonication at 20 kHz for a period of 5 min (SONICS Vibra Cell VC 455, 230 V, Germany). Debris were removed by centrifugation at 10,000 rev/min, at 4 C for 30 min. Step 2: Fractionation with ammonium sulfate. Solid ammonium sulfate was gradually added to the crude extract until reaching 35% saturation. The precipitate was removed by centrifugation at 10,000 rev/min, at 4 C for 25 min and the supernatant was continuously added solid ammonium sulfate to 70% saturation and stirred at 4 C for 4 h. After centrifugation, the precipitate was dissolved in 20 ml column buffer containing 1 M ammonium sulfate and the mixture was dialysed against 2 l of the same buffer for 12 h. Step 3: Phenyl-Sepharose hydrophobic interaction chromatography. The dialyzed enzyme solution was applied to a Phenyl-Sepharose column (1.610 cm) preequilibrated with 50 mM Tris–HCl buffer, pH 8.0 containing 1 M ammonium sulfate. After the column was washed with the column buffer containing 1 and 0.2 M ammonium sulfate respectively, the enzyme was eluted with distilled water at a flow rate of 1 ml/min. The elution peak was pooled for the next step. Step 4: Sephacryl S-200 size-exclusion chromatography. After being concentrated by ultrafiltration with a PM30 membrane (Amicon, USA), the enzyme solution was loaded onto a Sephacryl S-200 column (1.6100 cm) pre-equilibrated with column buffer. The enzyme was eluted at a flow rate of 0.5 ml/min with the same buffer and the active fraction was collected and concentrated. The purified enzyme was stored at 4 C. SDS-PAGE and native PAGE analysis Protein purity was analysed on 10% SDS/PAGE by the method of Laemmli (1970). The polymeric form of the enzyme was detected on 7.5% native PAGE with BSA and transferrin used as standards (Weber & Osborn 1969).
Purification, enzymatic properties of a recombinant D-hydantoinase
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Figure 1. The primary structure (479 amino acid residues) of the D-hydantoinase from Pseudomonas putida YZ-26 (The nucleotide sequence for the enzyme was deposited in GenBank under accession No. AY 387829).
Determination of molecular mass The subunit molecular mass (Mr) of the enzyme was estimated by SDS-PAGE. The molecular mass of the purified enzyme was determined by both native-PAGE and size-exclusion chromatography. A Pre-packed Superose 12 column was equilibrated with 50 mM Tris– HCl, 100 mM NaCl buffer (pH 8.0) on an AKTA purifier. The enzyme and standard proteins were individually loaded on the column and eluted with the same buffer at a constant flow rate of 1 ml/min at 25 C. The retention time of elution peaks was measured at 280 nm. Protein standards used were indicated in Figure 3. The apparent Mr of the purified enzyme was calculated from a plot of Kav against the logarithm of standard protein Mr values.
Results and discussion Expression of the D-hydantoinase Based on our previous work (Shi et al. 2005), the amino acid sequence of the D-hydantoinase was listed in Figure 1. The strain pE-HDT/E. coli BL21 could express the D-hydantoinase as the soluble form in LB medium. The enzyme activity of cultured cells reached 5.2–6.5 IU/ml culture at a cell turbidity of 10 at 600 nm, which was higher than that reported from other D-hydantoinases (Chien et al. 1998; Xu et al. 2003). It was shown from a SDS-PAGE analysis of expressed products that the subunit Mr of the enzyme was 52 kDa (Figure 2), the same as the value calculated from its amino acid sequence. By searching protein database with the BLAST program on the Internet, the enzyme had 90.0% amino acid homology with the D-hydantoinase from Pseudomonas putida CCRC 12857, while the homology with those from other bacterial strains was only 40–50% (Cheon et al. 2002). Purification of the D-hydantoinase The objective of developing a rapid and efficient purification process is to obtain homogeneous D-hydantoinase
without any affinity tags. Based on the experimental optimization of various matrix and separation conditions, a three-step purification process was finally selected, which combined ammonium sulfate fractionation, Phenyl–Sepharose hydrophobic interaction chromatography, and Sephacryl S-200 size-exclusion chromatography. The purification summary of the enzyme in Table 1 indicates that the purification factor is about 3.4-fold and the activity recovery reaches about 54.4%. The purity of D-hydantoinase is over 95% as estimated from SDS-PAGE analysis (Figure 2 Lane 4) and the specific activity for substrate DL-hydantoin reaches 48 IU/min/mg protein. Used as a routine procedure in labor reaches, the process of protein purification should be simple, efficient and with a high yield. The purification procedure described above for D-hydantoinase, either activity recovery or specific activity is in compliance with these requirements. In the first step, ammonium sulfate fractionation gives a reasonable amount of target protein and activity recovery. Hydrophobic interaction chromatography as a main step has a high performance in the separation efficiency. With the hydrophobic column, the target enzyme is highly concentrated in the fraction eluted with distilled water (Figure 2, Lane 3). Although the gel filtration has some dilution effect on the sample, the enzymatic activity recovery still reaches the ideal value after ultrafiltration (Figure 2 Lane 4).
Figure 2. SDS-PAGE analysis of expression and purification of the D-hydantoinase. Lane 1, Expression products of E. coli BL21/pEHDT; Lane 2, Supernatant of cells mixture after disruption and centrifugation; Lane 3, The fraction eluted with water from PhenylSepharose; Lane 4, Elution peak from Sephacryl-200 column. (D-hydantoinase band was indicated by an arrow).
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Figure 3. Molecular mass determination of the D-hydantoinase by size-exclusion chromatography (left below) and 7.5% (w/v) nativePAGE (right upper). Left below: D-hydantoinase and standard proteins were individually run on a pre-packed Superose 12 column equilibrated with 50 mM Tris–HCl, pH 8.0, 100 mM NaCl. The Kav is defined as (Ve)Vo)/(Vi)Vo), here Ve, Vi, Vo being the elution volume, the column volume (23.56 ml) and the void volume (7.86 ml), respectively. Proteins standards used (Mr and corresponding Kav in bracket) are (a) trypsinogen (24 kDa, 0.467); (b) alcohol dehydrogenase (41 kDa, 0.369); (c) albumin chicken egg (45 kDa, 0.361); (d) bovine serum albumin (dimer 136 kDa, 0.265); (e) transferrin (dimer 160 kDa, 0.226). The arrow points to the value corresponding to the D-hydantoinase (102.6 kDa, 0.281). Right upper: Lane 1, Bovine serum albumin (monomer-68, dimmer-136, trimer-204 kDa); Lane 2 and 3, the Purified D-hydantoinase; Lane 4, Transferrin dimer (160 kDa). The enzyme band is indicated by an arrow.
Physicochemical properties of the D-hydantoinase Molecular mass of the holoenzyme and its subunit It was reported that D-hydantoinases from various organisms usually form a homologous dimer or tetramer as the functional unit. (Abendroth et al. 2002; Xu et al. 2003). However, their subunit Mr values are quite different (Syldatk et al. 1999). The subunit and holoenzyme Mr values of the D-hydantoinase we purified were 52 and 103 kDa as determined by SDS-PAGE (Figure 2, Lane 4) and native-PAGE (Figure 3, on the right upper corner) respectively. In order to further examine the holoenzyme Mr, a size-exclusion chromatography was performed as described in Materials and methods. It indicates from the plot of Kav against the logarithm of standard protein Mr values in Figure 3 that the holoenzyme Mr should be 102.6 kDa. Therefore, the purified D-hydantoinase exists as a homologous dimer. Effect of pH and temperature on enzymatic activity The effects of pH and temperature on the D-hydantoinase activity were determined using the purified
enzyme with hydantoin as the substrate. The enzymatic activities at 100 mM buffer concentration with various pH values (KH2PO4–Na2B4O7 pH 6.0–9.0, NaOH– H3BO3 pH 8.5–10.0, NaHCO3–NaOH pH 9.5–11.0, KCl–NaOH pH 12–13) were measured. The optimal pH is found at 9.5. The general trend observed is that the more basic the buffer, the higher activity the enzyme. The enzymatic activity is completely lost under pH 6.0, in contrast, it maintains nearly 60% activity at a pH even more than pH 12 (Figure 4). The high enzymatic activity at the alkaline condition confers significant advantages for the industrial bioconversion of amino acids. After the enzyme solution had been incubated at different temperatures ranging from 25 to 70 C for 30 min, the enzymatic activity was measured under the standard assay condition. The optimal temperature for the enzyme is 45 C and it is only stable at a temperature below 50 C (Figure 5). These results are in accordance with those reported for D-hydantoinases from other Pseudomonas species (Takahashi et al. 1978). Substrate specificity and kinetic parameters The substrate specificity of the enzyme was tested with various substrate compounds, such as hydantoin, dihydrourarcil, phenylhydantoin and succinimide. The highest activity was observed when dihydrouracil was used as the substrate (Table 2). It was shown from kinetic parameters that Km and Vmax values were 0.20 mM and 0.30 mmole/min/mg protein when DL-hydantoin was used as the substrate, while Km and Vmax values were 0.02 mM and 5.30 mmol/min/mg protein when dihydrouracil used as its substrate. These results indicate that the optimal substrate for the enzyme is dihydrouracil, not DL-hydantoin, as observed in most D-hydantoinases from microbial sources (Takahashi et al. 1978; Kikugawa et al. 1994) Effect of divalent metal ions on enzyme activity As can be seen in Table 3, divalent metal ions at 1 mM concentration obviously affected the enzymatic activity. The additional extrinsic Co2+, Mn2+ and Ni2+ ions enhanced the enzymatic activity up to 179%, 174% and 154% respectively, while Zn2+ and Cd2+ ions attenuated the enzymatic activity down to 68% or 44% respectively and Cu2+ completely inhibited its activity. From above results, it is likely that Co2+, Mn2+ or Ni2+ ions facilitate the induction of a favorable enzyme conformation and coordinate the combination of enzyme and substrate, while Zn2+ or Cd2+ ions may induce the dissociation of the enzyme molecule and
Table 1. A purification summary of the D-Hydantionase (starting from 200 ml culture). Purification steps
Total proteins (mg)
Total activity (Units)
Activity recovery (%)
Specific activity (Units/mg)
Purification (-fold)
Crude extract (NH4)2SO4 Fract. Phenyl Sepharose Sephacryl S-200
61.8 48.8 15.5 9.8
881.8 767.7 575.6 476.7
100.0 87.8 64.6 54.4
14.3 15.7 37.1 48.5
1.0 1.1 2.6 3.4
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Purification, enzymatic properties of a recombinant D-hydantoinase Optimum pH pH stability
Relative activity(%)
100 80 60 40 20 0
6
7
8
9
9.5
10
11
12
pH Figure 4. Effect of pH on the D-hydantoinase acitivity. About 100 mM various buffers were used as follows: Na2HPO4-Citric acid buffer, pH 4.0–6.0; KH2PO4–Na2B4O7 buffer, pH 6.5–9.0; NaHCO3–NaOH buffer, pH 9.5–11.0; KCl–NaOH buffer, pH 12–13. The relative activity was expressed as a percentage of the maximum activity assayed under the experimental conditions. All data were the average of at least three tests.
Cu2+as a heavy metal ion may lead to the aggregation of the enzyme (also see discussion below).
Relative activity(%)
Dissociation of the D-hydantoinase by zinc ion In order to further explore the dissociation of the enzyme by Zn2+ ion, the enzyme solution was gradually mixed with different concentrations (from 0.01 to 5 mM) of extrinsic Zn2+ ion and incubated at 37 C for 2 h. Then the co-relation between enzymatic activity and molecular mass was evaluated by both native-PAGE and size-exclusion chromatography. Results shown in Figure 6 indicate that there is no change in either the molecular mass or the enzymatic activity at a Zn2+ concentration below 0.5 mM, while the activity is lowered to about 60–70% at 1 mM Zn2+, accompanied with the appearance of monomer molecule. At a Zn2+ concentration above 2 mM, the enzymatic activity is completely lost and the enzyme is dissociated into its monomer. A possible explanation of above results is that in the first state, the lower concentration of Zn2+ is not enough to change the charge transfer of the enzyme,
100 90 80 70 60 50 40 30 20 10 0
Thermostability Optimum Temp.
maintaining the correct protein conformation; in the second state, the amount of Zn2+ changes charge– charge interaction and charge transfer at the active site of the enzyme, resulting in the low activity and enzyme dissociation; and in the third state, the high concentration of Zn2+ not only changes charge–charge interaction and charge transfer at the active site, but also surface charges of the enzyme molecule, leading to enzyme aggregation and inactiviation. It has been reported that certain hydrolases require Zn2+ ion as the modulator to induce the enzyme conformation and to coordinate substrate hydrolysis, such as urease, hydantoinase, dihydropyrimidinase etc. (Kim & Kim 1998; May et al. 1998). However, the D-hydantoinase we have purified seems to be an exception. The effect of Zn2+ ion on this enzyme is similar to D-aminoacylase with regard to their conformation and active site residues. Similar to D-aminoacylase, the enzyme belongs to TIM barrel metal-dependent hydrolase which has ab-binuclear subset to bind essential metal ions (Holm & Sander 1997). Therefore, Co2+, Mn2+ and Ni2+ ions as activators may tightly bind to the b-subsite to enhance the enzyme activity, but Zn2+ ion as the inhibitor may hold the a-subsite to attenuate the activity
Table 2. Substrate specificities of the D-hydantoinase.
25
35
40
45
50
55
60
Substrates
Activity (U/mg)
Hydantoin (100 mM) DL-5-Phenylhydantoin (10 mM) DL-p-Hydroxyphenylhydantoin (10 mM) Dihydrouracil (50 mM) Uracil (25 mM) Succinimide (25 mM)
48.5 10.65 11.84 600.85 None None
65
Temperature Figure 5. Effect of temperature on the D-hydantoinase activity. The optimal temperature of the D-hydantoinase was conducted in 50 mM Tris–HCl (pH 8.0) at the definite temperature when DL-hydantoin was used as the substrate. The thermostability was determined by incubating the D-hydantoinase at various temperatures for 30 min and then an appropriate amount of the incubated mixture was removed to assay the activity. All data were the average of at least three measurements.
Table 3. Effect of divalent metal ions on the D-Hydantoinase activity. Metal ions None Zn2+ Mn2+ Cu2+ Cd2+ Ni2+ Co2+ Ca2+ Mg2+ 1 2 3 4 5 6 7 8 9 (1 mM) Relative 100 activity (%)
68
174
0.4
44
154
179
97
120
680
Figure 6. Dissociation of the D-hydantoinase in the presence of Zn2+. Left: Size–exclusion chromatography: a AKATA purifier and a prepacked Superose 12 column (10/30) were equipped. The reaction mixture of the enzyme incubated for 2 h at room temperature was loaded on the column. The elution buffer was 20 mM Tris–HCl buffer containing 0.1 M NaCl, (pH 8.0) at a flow rate of 1 ml/min. Purple trace: D-hydantoniase dimer; Pink trace: D-hydantoinase with 0.5 mM Zn2+; Green trace: D-hydantoinase with 1 mM Zn2+. Right: 8% Native-PAGE analysis: The Arabic numbers indicate the concentration of Zn2+ (mM) in the reaction mixture of the enzyme which was incubated for 2 h at RT.
(Lai et al. 2004). It must be emphasized that Zn2+ as the second-most abundant transition metal in living organisms after iron plays an important role in physiological activity and enzyme as the biocatalyst is the main power of metabolism. Therefore, the research of Zn2+ function in enzyme molecules should not be neglected.
Acknowledgements This project was supported by the National Science Foundation of Shanxi province (NSFSX, 031042), P.R. China. We also thank Dr Tao Yuan, Sanofi Pasteur, in Canada for his valuable discussion.
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