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CURRENT MICROBIOLOGY Vol. 55 (2007), pp. 61–64 DOI: 10.1007/s00284-005-0455-6 Current Microbiology An International Journal ª Springer Science+Business Media, LLC 2007 Gene Cloning, Expression, and Substrate Specificity of an Imidase from the Strain Pseudomonas putida YZ-26 Ya-wei Shi, Li-fang Cui, Jing-ming Yuan Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of the National Ministry of Education, Shanxi University, 92 Wucheng Road, Taiyuan, Shanxi 030006, PRC Received: 22 November 2005 / Accepted: 30 June 2006 Abstract. A gene-encoding imidase was isolated from Pseudomonas putdia YZ-26 genomic DNA using a combination of polymerase chain reaction and activity screening the recombinant. Analysis of the nucleotide sequence revealed that an open reading frame (ORF) of 879 bp encoded a protein of 293 amino acids with a calculated molecular weight of 33712.6 kDa. The deduced amino-acid sequence showed 78% identity with the imidase from Alcaligenes eutrophus 112R4 and 80% identity with Nterminal 20 amino-acid imidase from Blastobacter sp. A17p-4. Next, the ORF was subcloned into vector pET32a to form recombinant plasmid pEI. The enzyme was overexpressed in Escherichia coli and purified to homogeneity by Ni2+–NTA column, with 75% activity recovery. The subunit molecular mass of the recombinant imidase as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis was approximately 36 kDa, whereas its functional unit was approximately 141 kDa with four identical subunits determined by size-exclusion chromatography. The purified enzyme showed the highest activity and affinity toward succinimide, and some other substrates, such as dihydrouracil, hydantoin, succinimide, and maleimde, were investigated. Imidases from bacteria are attracting increasing attention as novel agents for the production of useful organic acids as well as new tools for fine enzymatic synthesis of chiral compounds. These include unnatural amino acid [1], pyruvate [2], and 3-carbamoyl-alpha-picolinic acid [3], all of which are critical building blocks for semisynthetic antibiotics, pesticides, and food additives. Imidase—which is also known as dihydropyrimidinase, hydantoinase, dihydropyrimidinase, and amidohydrolase because of its bound-substrate specificity [4, 5]—has been shown to participate in pyrimidine metabolism in vivo or in bioconversion of organic acids in vitro [6, 7]. Most of the imide-hydrolyzing enzymes share limited sequence homology and have different substrate charts. In general, they are dimer or tetramer with a 50- to 60kD subunit [4, 5], with the exception of two imidases being reported so far as having a lower subunit molecular mass of 30 to 40 kDa. Contrary to known imidase, Correspondence to: Y.-W. Shi; email: [email protected] an imidase with a 35-kD subunit from Blastobacter sp. A17p-4, which was first purified and characterized by Ogawa et al., preferably hydrolyses cyclic imides and does not accept 5¢-monosubstituted hydantoins as substrate [8, 9]. The imidase gene from the strain Alcaligenes eatrophus 112R4 was first isolated and expressed in Escherichia coli [9]. In bacteria, imidase is involved in ring-opening hydrolysis of cyclic imides to half-amides, and the resulting half-amide is hydrolyzed by halfamidase to dicarboxylates, which then undergoes further transformation through tricarboxylic acid (TCA) cyclic reactions [8]. On the basis of our work on D-hydantoinase from the strain P. putdia YZ-26, a third imidase with low subunit molecular weight is reported herein. Materials and Methods Bacterial strains, plasmids, and medium. The P. putdia strain YZ26 used was screened and identified by our laboratory. E. coli was used as host in the cloning and expressing procedures. The thioredoxindeleted pET32a (Novagen) was used as expression vector. pUC118 and 62 polymerase chain reaction (PCR) primers were purchased from TaKaRa (Dalian Branch, China). YCG medium (0.5% yeast, 0.5% peptone, 0.5% glycerol, 0.2% K2HPO4, and 0.1% DL-hydantoin, adjusted to pH 7.0) was used as screening medium for activity assay on microtiter plate. The recombinant strain pEI/E. coli BL21 was employed for expressing the enzyme in Lura-Bertani (LB) medium. Screening the imidase. Genomic DNA from P. putdia YZ-26 was isolated according to the method of Lewinton [10] and digested with EcoRI for 6 hours at 37C. Approximately 2 to 9 kb DNA fragments recovered from agarose gel were ligated into the corresponding site of pUC118 and transformed into E. coli JM109. The transformants were initially screened on LB agar plate containing X-gal, IPTG, and 100lg /mL ampicillin. White transformants were picked out and inoculated into a microtiter plate (96 wells) containing 50 ll liquid YCG medium for each well and incubated at 37C overnight. Afterward, imidase activity per well was detected by a colorimetric method using DLhydantoin as substrate [11]. Positive recombinants in wells developed an unequivocal yellow colour, whereas negative recombinants produced a reddish colour or were colourless. DNA sequence analysis and subcloning. The imidase gene DNA sequence of a positive recombinant (pUS804) was analyzed by TaKaRa. The deduced amino-acid sequence of the open reading frame (ORF) was aligned using the ProSite database at ExPASY. The ORF of imidase from pUS804 was reamplified by PCR using forward primer (5¢-CGGAATTCATGGCCAAGGAAATC-3¢) and reverse primer (5¢-CCGAAGCTTTCACTTCTTGCGCGG-3¢). Additional restriction sites for EcoRI and HindIII were introduced for subsequent cloning of the gene. The amplification was performed with 30 cycles at 94C for 1 minute, 50C for 1 minute, and 72C for 1 minute, and extension for 10 minutes by using a PTC-200 Peltier Thermal Cycler. The amplified fragment, digested with EcoRI and HindIII, was inserted into the corresponding sites of the modified vector pET32a to form recombinant plasmid pEI. Expression and purification of imidase. Recombinant plasmid was transformed into E. coli BL21 (DE3) host cells for large-scale protein production. The host cells containing the recombinant plasmid were cultured in LB medium supplemented with 100 lg/mL ampicillin on a shaker at a speed of 170 rpm at 37C. When the optical density (A600nm) of cells reached approximately 0.6 to 0.8, 0.2 5mM IPTG was added, and the cells were continuously incubated at 37C for 4 hours under the same conditions to induce imidase production. Cells were harvested by centrifugation at 4,000 rpm for 15 minutes, and the pellet was suspended in 50 mM Tris-HCl (pH 8.0) containing 500 mM NaCl and further disrupted by ultrasonication (SONICS Vibra Cell VC 455, Germany). After centrifugation at 16,000 rpm (Sorvall SS-34 rotor) for 30 minutes at 4C, the supernatant was applied to an Ni2+-NTA affinity column and washed thoroughly with binding buffer (50 mM Tris-HCl [pH 8.0] 500 mM NaCl, and 50 mM imidazole). Then the target protein was eluted out with elution buffer (50 mM Tris-HCl [pH 8.0] containing 500 mM NaCl and 1 M imidazole). The enzyme was collected and stored at 4C for further analysis. Assay of imidase activity. Imidase activity was measured by a spectrometric method using DL-hydantoin as substrate [11]. The reaction rate was determined by monitoring the absorbance change at 430 nm and the concentration of the product N-carbamyl-glycine was determined from a standard calibration plot. In the case of succinimide or maleimide being used as substrate, high-performance liquid chromatography (HPLC) was used to determine imidase activity. An appropriate amount of imidase was mixed with 20 mM succinimide or maleimide dissolved in 100 mM Tris-HCl (pH 8.0) or in 50 mM CURRENT MICROBIOLOGY Vol. 55 (2007) phosphate buffer pH 6.5, respectively, at a total volume of 200 ll. The reaction mixture was incubated at 37C for 10 minutes and stopped by boiling for 5 minutes. After centrifugation, the supernatant was analyzed on an HPLC system (Waters) equipped with a Symmetry C18 column (4.6 mm x 150 mm, 5 lm). The mobile phase used in this process was a mixture of methanol and 50 mM phosphate buffer (pH 6.5) (5:95 v/v) at a flow rate of 1.0 ml/min. The ultraviolet detector was set at 210 nm for succinimide and at 255 nm for maleimide. One unit of enzyme was defined as the amount of the enzyme that catalyzes substrate to form the product at a rate of 1 lmol /min under the assay conditions described previously. Molecular mass of the enzyme. The apparent molecular mass of the subunit was estimated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) [12]. The native molecular mass of the imidase was determined by size-exclusion chromatography with a prepacked Superose12 (10/30) column pre-equilibrated and eluted with 50 mM Tirs-HCl and 100 mM NaCl (pH 8.0). The column was calibrated by standard proteins, including trypsinogen (24 kDa), alcohol dehydrogenase (41 kDa), egg albumin (45 kDa), bovine serum albumin dimer (136 kDa), and transferrin dimer (160 kDa). The native molecular mass of the enzyme was calculated from Kav plot against the logarithm of standard protein. Results and Discussion Cloning imidase gene from the strain P. putdia YZ26. Genomic DNA extracted from P. putdia YZ26 was randomly cut by EcoRI, and fragments with a length of approximately 2 to 9 kb recovered from agarose gel were inserted into vector pUC118 and then transformed into E. coli JM109. By hydantoinase activity assay of approximately 2000 colonies on a 96well microtiter plate, 1 positive colony with higher hydantoinase activity was selected. The plasmid extracted from this colony was designated pUS804, which contained a 2.69-kb inserted fragment (GenBank accession no. DQ093858). DNA sequence analysis showed that an ORF of 879 bp was involved in this fragment, which corresponded to 293 amino acids with a calculated molecular mass of 33712.6 kDa (Fig. 1). The deduced amino-acid sequence showed 78% identity with the imidase (291 amino acids) from A. eatrophus 112R4 [9] and 80% identity with the 20 N-terminal amino acids of imidase from Blastobacter sp. A17p-4 [8]. However, no similarity was found compared with any known 50- to 60-kDa imide-hydrolyzing enzymes on the protein sequence database available from the Internet using the BLAST search engine [13]. To express the gene, the ORF was reamplified by PCR with pUS804 as template, and the resultant PCR product was inserted into the modified pET32a to form recombinant plasmid pEI. After being introduced into E. coli BL21, the strain pEI/E. coli BL21 expressed the active imidase in LB medium, resulting in a dense band of 36 kDa on SDS-PAGE (see next section). Y.-W. Shi et al.: Imidase Gene Cloning, Expression, and Substrate Specificity 63 Fig. 2. SDS-PAGE analysis of expression product and imidase purification. Lane 1 = Culture of uninduced cells. Lane 2 = Culture of induced cells. Lane 3 = Supernatant after sonication and centrifugation. Lane 4 = Sample eluted from Ni2+-NTA column. Fig. 1. DNA sequence analysis of 2.69 kb fragment from P. putida YZ-26 genomic DNA. The ORF was shown to be 1282 bp to 2163 bp and the corresponding amino acids were deduced. This sequence was deposited to GenBank under accession no. DQ093858. Expression and purification of imidase. Recombinant E. coli BL21(pEI) could express imidase in soluble form, and the activity reached 5.19 U/ml cultured cell as described in Materials and Methods. (His)6-tagged imidase was subsequently purified using an Ni2+-NTA agarose, which occurred as a single band with an apparent molecular mass of 36 kDa as determined by SDS-PAGE (Fig. 2). Overall recovery of the enzymatic activity by one-step affinity purification was 75% (Table 1). Molecular mass and form of imidase. The subunit molecular mass of the (His)6-tagged imidase was approximately 36 kDa (Fig. 2). It has been reported that the functional unit of imidase from divergent sources usually occurs as dimer or tetramer [4, 5]. As such, size-exclusion chromatography was performed to evaluate the molecular form of the native imidase on an AKTA purifier. The result indicated that the retention volume of the native enzyme (11.75 ml) is close to the that of the bovine serum albumin (BSA) dimer (12.02 ml). Using the standard curve–deduced molecular mass curve of standard proteins (Fig. 3), the molecular mass of the native imidase was estimated to be 141 kDa. By contrast to the trimer imidase from Blastobacter sp. strain A17p-4 [8], the imidase isolated in the current study corresponds to a tetramer of identical subunits. Substrate specificity of imidase. Substrate specificity of the imidase was tested with various compounds, such as hydantoin, dihydrourarcil, succinimide, and maleimide. Imidase demonstrated the highest activity toward succinimide, which is in accordance to the kinetic parameters listed in Table 2. Imidase demonstrated 100- to 500-fold higher velocity in the hydrolysis of simple cyclic imide (e.g., succinimide and maleimide) than in the hydrolysis of cyclic ureides (e.g., hydantoin or dihydrourarcil). However, the optimal substrate of most known imidases with 50- to 60-kDa subunits is 5¢-substitute hydantoin or dihydrouracil, not succimide. Similar results have also been observed in imidase from Blastobacter sp. strain A17p-4 [8] and from A. eatrophus 112R4 [9]. This implies that the conformation of the active site of the imidase may prefer the simple cyclic imide. Conclusion Just as D-hydantoinase with cyclic ureide as substrate was reported in our previous work [14, 15], another imidase with succinimide as optimal substrate was achieved in the same strain, P. putida YZ-26. With respect to substrate specificity, kinetic parameters, subunit molecular mass, and amino-acid sequence, this enzyme is different from approximately 50- to 60-kDa imidases, such as D-hydantoinase, which is the third imidase found so far in bacteria. Our data demonstrate that two different kinds of imidases can be present in Pseudomonas as reported in Blastobacter spp. [7]. 64 CURRENT MICROBIOLOGY Vol. 55 (2007) Table 1. Purification summary of the recombinant imidase (1 L culture) Steps Total protein (mg) Total activity (lmol/min) Specific activity (lmol/min/mg) Yield % Fold of purification Extract Ni2+—NTA 331.1 24.7 988.6 744.3 2.99 30.1 100 75,3 1.0 10.1 Literature Cited Fig. 3. Determination of the molecular mass of the imidase by sizeexclusion chromatography. Imidase and standard proteins were applied to a prepacked Sepharose 12 column, pre-equilibrated in 50 mM TrisHCl (pH 8.0) and 100 mM NaCl. Kav is defined as (Ve-Vo/Vi-Vo) with Ve, Vi, and Vo as the elution volumes, the column volume (23.56 ml), and the void volume (7.86 ml), respectively. Protein standards (with Mr and corresponding Kav in bracket) are (a) trypsinogen (24 kDa, 0.467), (b) alcohol dehydrogenase (41 kDa, 0.369), (c) chicken egg albumin (45 kDa, 0.336) (d) bovine serum albumin (dimer 136 kDa, 0.265, and (e) transferrin (dimer 160 kDa, 0.226). The arrow points to the value corresponding to the imidase (140 kDa, 0.248). Table 2. Substrate specificity of the recombinant imidase Substrates km (mM) kcat (min)1) kcat/km (mM)1min)1) Hydantoin (100 mM) Dihydrouracil (100 mM) DL-5-Phenylhydantion (10 mM) DL-p-Hydroxyphenylhydantion (10 mM) Succinimide (20 mM) Maleimide (20 mM) 53.5 74.3 — — 126.4 548.6 — — 2.4 7.3 — — 29.2 39.1 89641.0 28750.4 3061.4 735.5 ACKNOWLEDGMENTS This research was supported by the grant from National Science Foundation of Shanxi Province (NSFSX, 031042), PRC. 1. 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