l -Asparaginase from Erwinia Chrysanthemi 3937: Cloning, expression and characterization

Journal of Biotechnology 127 (2007) 657–669

l-Asparaginase from Erwinia Chrysanthemi 3937: Cloning, expression and characterization Georgia A. Kotzia, Nikolaos E. Labrou ∗ Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece Received 16 December 2005; received in revised form 19 May 2006; accepted 31 July 2006

Abstract Bacterial l-asparaginases (l-ASNases) catalyze the conversion of l-asparagine to l-aspartate and ammonia. In the present work, we report the cloning and expression of l-asparaginase from Erwinia chrysanthemi 3937 (Erl-ASNase) in Escherichia coli BL21(DE3)pLysS. The enzyme was purified to homogeneity in a single-step procedure involving cation exchange chromatography on an S-Sepharose FF column. The enzymatic and structural properties of the recombinant enzyme were investigated and the kinetic parameters (Km , kcat ) for a number of substrates were determined. In addition, we found that the enzyme can be efficiently immobilized on epoxy-activated Sepharose CL-6B. The immobilized enzyme retains most of its activity (60%) and shows high stability at 4 ◦ C. The approach offers the possibility of designing an Erl-ASNase bioreactor that can be operated over a long period of time with high efficiency, which can be used in leukaemia therapy. © 2006 Elsevier B.V. All rights reserved. Keywords: l-Asparaginase; Hydrolase; Enzyme immobilization; Leukaemia

1. Introduction l-ASNase (l-asparagine amidohydrolase, E.C. 3.5.1.1) is used for the effective treatment of acute lymphoblastic leukaemia (ALL) (Wriston, 1985; Goward Abbreviations: ALL, acute lymphoblastic leukaemia; ErlASNase, l-asparaginase from Erwinia chrysanthemi; GDH, glutamate dehydrogenase; ORF, open reading frame; PAGE, polyacryamide gel electrophoresis; SDS, sodium dodecyl sulfate; Suc, succinic acid ∗ Corresponding author. Fax: +30 210 5294308. E-mail address: [email protected] (N.E. Labrou). 0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.07.037

et al., 1992). It catalyzes the conversion of l-Asn to l-Asp and ammonia. The anti-tumour activity of the enzyme is based on the dependence of certain tumour cells on an extracellular supply of l-Asn. Unlike normal cells, malignant cells can only synthesize l-Asn slowly, due to their deficiency in l-asparagine synthetase (Keating et al., 1993; Moola et al., 1994). Thus, depletion of the circulating pools of l-Asn by l-ASNase leads to the destruction of the tumour cells, since they are unable to complete protein synthesis. The functional form of l-ASNase exists as a tetramer of identical subunits, with molecular mass

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in the range of 140–160 kDa (Aung et al., 2000; Aghaiypour et al., 2001a,b; Kozak et al., 2002). Each of the four active sites is located between the N- and C-terminal domains of two adjacent monomers. Thus, the l-ASNase tetramer can be treated as a dimer of dimers. Despite this fact, the active enzyme is always a tetramer (Swain et al., 1993; Khushoo et al., 2004). l-ASNases from Erwinia chrysanthemi and Escherichia coli are currently in clinical use as effective drugs in the treatment of ALL. They are also used in the treatment of Hodgkin’s disease, acute myelocytic leukaemia, acute myelomonocytic leukaemia, chronic lymphocytic leukaemia, lymphosarcoma, reticulosarcoma and melanosarcoma (Stecher et al., 1999; Duval et al., 2002). Unfortunately, despite the wide use of l-ASNases, the therapeutic response of patients rarely occurs without some evidence of toxicity. The main side effects of l-ASNases are: liver dysfunction, pancreatitis, diabetes, leucopoenia, neurological seizures, and coagulation abnormalities that may lead to intracranial thrombosis or haemorrhage (Duval et al., 2002). Another limiting factor of l-ASNase treatment is the development of hypersensitivity, which ranges from mild allergic reactions to anaphylactic shock (Moola et al., 1994). Because the l-ASNases from E. coli and Er. chrysanthemi are immunologically distinct, they provide an important alternative therapy to patients who become hypersensitive to one of the enzymes (Moola et al., 1994). With a view to characterize enzymes with less toxic side effects, several members of a larger family of homologous l-ASNases have been thoroughly investigated over many years (Boyse et al., 1967; Ehrman et al., 1971; Cammack et al., 1972; Wriston and Yellin, 1973; Krasotkina et al., 2004; Kotzia and Labrou, 2005). In addition, because the anti-tumour activity of l-ASNase is also a function of its halflife in the blood (Fernandes and Gregoriadis, 1997), attempts have been made to increase the half-life, for example by entrapment of the enzyme in liposomes (Neerunjun and Gregoriadis, 1976) or microcapsules (Chang, 1984), and by covalent coupling to macromolecules such as dextran (Wileman et al., 1986), albumin (Poznansky et al., 1982) or monomethoxypolyethylene glycol (mPEG) (Kamisaki et al., 1982) which is on the market. Unfortunately, none of these approaches have managed to eliminate the disadvan-

tages of l-ASNase treatment, leaving scientists with the need to identify and characterize new enzymes with better properties. In the present work, we describe the cloning, expression, and kinetic characterization of a new l-ASNase from Er. chrysanthemi, strain 3937.

2. Materials l-Asn and l-Gln were obtained from Serva (Heidelberg, Germany). 1,4-Butanediol diglycidyl ether, ␣ketoglutaric acid and Sepharose CL-6B were obtained from Sigma Co. (St. Louis, USA). N␣ -Acetyl-l-Asn and succinamic acid were obtained from SigmaAldrich (Milwaukee, USA). NADH (disodium salt, grade II, ca. 98%) and crystalline bovine serum albumin (fraction V) were purchased from Boehringer Mannheim (Germany). Nessler’s reagent, ␤-alanine amide and glutamate dehydrogenase (GDH) were obtained from Fluka (Taufkirchen, Germany) and ninhydrin from Merck (Darmstadt, Germany). TOPO cloning kit and all other molecular biology reagents were from Invitrogen (USA).

3. Methods 3.1. Cloning, expression, and purification of l-ASNase Er. chrysanthemi 3937 was grown at 30 ◦ C in a medium containing 1% peptone, 0.5% yeast extract and 1% NaCl. After 18 h, cells were pelleted by centrifugation and genomic DNA was isolated according to a standard procedure (Sambrook et al., 1989). A BLASTN search of the Er. chrysanthemi 3937 genome sequence (Sanger Centre database) using as a query the nucleotide sequence encoding l-ASNase from Er. chrysanthemi NCPPB 1125 (CAA31239) yielded a single 1047-bp ORF that was supposed to encode for l-ASNase. PCR was used to amplify the full-length ORF from genomic DNA using oligo primers synthesized to the 5 region of the gene from the ATG start codon (5 -ATG GAA CGA TGG TTT AAA TCT CTG-3 ) to the 3 end of the gene, including the TGA stop codon (5 -TCA ATA GGT GTG GAA ATA GTC CTG G-3 ). The PCR reaction was carried out in a total volume of 50 ␮l containing 55 ng of each primer,

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10 ng template genomic DNA, 0.2 mM of each dNTP, 5 ␮l 10× Pfu buffer and 2 units of Pfu DNA polymerase (Stratagene, USA). The PCR procedure was composed of 30 cycles of 2.5 min at 96 ◦ C, 2 min at 46 ◦ C, and 3 min at 72 ◦ C. A final extension for 10 min at 72 ◦ C was carried out after the 30 cycles. The resulting PCR amplicon was TOPO ligated into a T7 expression vector (pCR® T7/CT-TOPO® ). The resulting expression construct was sequenced along both strands and was used to transform competent BL21(DE3)pLysS E. coli cells. E. coli cells harbouring plasmid were grown at 37 ◦ C in 1 l LB medium containing 100 ␮g/ml ampicillin and 34 ␮g/ml chloramphenicol. The synthesis of ErlASNase was induced by the addition of 1 mM IPTG when the absorbance at 600 nm was 0.6–0.8. Five hours after induction, cells were harvested by centrifugation at 10,000 × g (4 ◦ C) for 20 min, suspended in potassium phosphate buffer (20 mM, pH 5.5), sonicated, and centrifuged at 10,000 × g (4 ◦ C) for 5 min. The supernatant was collected and applied to a S-Sepharose FF (2 ml, 1.5 cm × 1.5 cm i.d.) column, previously equilibrated with potassium phosphate buffer (20 mM, pH 5.5). Non-adsorbed protein was washed off with 30 ml equilibration buffer followed by 30 ml of potassium phosphate buffer (20 mM, pH 7.5). Bound Erl-ASNase was eluted with potassium phosphate buffer (20 mM, pH 8.5). 3.2. Assay of enzyme activity and protein Enzyme assays were performed at 37 ◦ C using a Hitachi U-2000 double beam UV–VIS spectrophotometer equipped with a thermostated cell holder (10 mm pathlength). l-ASNase activities were measured by determining the rate of ammonia formation using glutamate dehydrogenase (Balcao et al., 2001). The final assay volume of 1 ml contained 71 mM Tris–HCl buffer, pH 8.0, 1 mM l-Asn, 0.15 mM ␣ketoglutaric acid, 0.15 mM NADH, 4 units glutamate dehydrogenase, and sample containing Erl-ASNase activity. Alternatively, the rate of ammonia formation was also measured at 37 ◦ C using Nessler’s reagent or ninhydrin reagent (Sheng et al., 1993). One unit of lASNase activity is defined as the amount of enzyme that liberates 1 ␮mol of ammonia from l-Asn per minute at 37 ◦ C. Protein concentration was determined at 25 ◦ C by the method of Bradford (1976) using bovine serum albumin (fraction V) as standard.

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3.3. Kinetic analysis Steady-state kinetic measurements were performed in 0.1 M Tris–HCl buffer, pH 8.0 (or pH 8.2 for l-Gln), by varying the concentration of the substrate (l-Asn, l-Gln, N␣ -Acetyl-l-Asn, succinamic acid, ␤-alanine amide) (Kelo et al., 2002). The kinetic parameters kcat and Km were calculated by non-linear regression analysis of experimental steady-state data. Turnover numbers were calculated on the basis of one active site per subunit. Kinetic data were analyzed using the computer program GraFit (Erithacus Software Ltd.) (Leatherbarrow, 1998). The dependence of the reaction rate on temperature was evaluated by measuring enzyme activity for l-Asn hydrolysis at different temperatures under the same conditions reported above. The data were analyzed by Arrhenius and Eyring plots as described in Section 4.3. 3.4. Bioinformatics analysis and molecular modeling The amino acid sequence of Erl-ASNase exhibits 91% sequence identity with the available Erl-ASNase crystal structure (PDB code 1hg0) (Aghaiypour et al., 2001b), and therefore molecular models of ErlASNase from Er. chrysanthemi 3937 were constructed. The models were constructed using MODELLER 6 (Sali and Blundell, 1993, http://www.infobiosud.cnrs. fr/bioserver) or using SWISS-MODEL (http://www. expasy.org/swissmod/). The crystal structures of Er. chrysanthemi in complex with succinic acid and lGln (PDB codes 1hg0 and 1hfw) were used as templates. Rigorous protein structure quality assessment was carried out using PROSA II (Sippl, 1993) and Verify 3D (Luthy et al., 1992). PROSA II and Verify 3D both yielded overall scores as well as local profiles which could be used to localize areas of unusual packing and/or solvent exposure characteristics. The overall scores were used to choose the final model. Analysis of packing, solvent exposure and stereochemical properties suggests the final Erl-ASNase models to be of good quality (PROSA II score 0.187; Verify 3D score 0.332). For inspection of models and crystal structures, the program PyMOL was used (DeLano, 2002). Sequences homologous to Erl-ASNase were sought in NCBI using BLAST (Altschul et al., 1990). The resulting sequence set was aligned with ClustalW

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(Thompson et al., 1994). ESPript (http://www.prodes. toulouse.inra.fr/ESPript/cgi-bin/ESPript.cgi) was used for alignment visualization and manipulation. Nonbonded interactions were analyzed by MolTalk (http://www.i.mol.talk.org). 3.5. Thermal stability of Erl-ASNase Thermal stability of Erl-ASNase was investigated at different pH values as follows: samples of the enzyme in 10 mM KH2 PO4 buffer (pH 5.5, 7 and 8.5), were incubated at different temperatures (35–65 ◦ C) for 7.5 min. Subsequently, the samples were assayed for residual activity using the coupled enzyme assay method described previously. The Tm value at each pH was determined from a plot of relative inactivation (%) versus temperature (◦ C). The Tm value is the temperature at which 50% of the initial enzyme activity is lost after heat treatment. The time course of thermal inactivation of ErlASNase was studied at 37 ◦ C and 41 ◦ C in 0.01 M potassium phosphate buffer pH 7.0. The rate of inactivation was measured by periodically removing samples for assay of enzymatic activity. Observed rates of inactivation (kin ) were deduced from plots of log (% of remaining activity) versus time (h). 3.6. Enzyme immobilization Sepharose CL-6B was first activated with 1,4butanediol diglycidyl ether to facilitate the immobilization of Erl-ASNase (Axarli et al., 2005). The procedure was carried out as follows: a solution of NaOH (0.6 M, 3 ml) was added to washed cross-linked agarose gel (Sepharose CL-6B, 1.5 g). The suspension was tumbled at room temperature for 5.5 h prior to adding 1,4-butanediol diglycidyl ether (1 ml) in the presence of 2.5 mg sodium borohydride. The reaction was left to proceed with slow agitation overnight at room temperature. After completion of the reaction, the activated gel was recovered by suction filtration and was washed, with ddH2 O (100 ml). The washed activated gel was divided into two equal parts. To the first part, a solution of Erl-ASNase (16.2 units in 1 ml of 0.1 M potassium phosphate buffer, pH 9) was added, while to the second part, a solution of Erl-ASNase (17.9 units in 1 ml of 0.1 M potassium phosphate buffer, pH 9) containing 5 mM succinic acid

was added. The reactions were allowed to proceed at 4 ◦ C for 22 h with slow agitation. After completion of the reaction, the gels were washed with 20 ml of 0.1 M potassium phosphate buffer (pH 9.0), followed by 20 ml of 50 mM Tris–Cl buffer (pH 8.2). The gels with the immobilized enzyme were stored at 4 ◦ C in 50 mM Tris–Cl buffer, pH 8.2. The amount of enzyme bound to Sepharose was determined as the difference between the initial and residual protein concentrations. Repeated washing steps with 50 mM Tris–Cl buffer, pH 8.2, lead to a negligible loss of activity. 3.7. Turnover of l-Asn with immobilized Erl-ASNase Immobilized Erl-ASNase (7.4 mU Erl-ASNase) was suspended in 1 ml of 50 mM Tris–Cl buffer pH 8.2, containing 8.5 mM l-Asn. The reaction mixture was subsequently left on rotary shaker at 25 ◦ C. At time intervals the reaction mixture was centrifuged to separate the reaction products from the immobilized biocatalyst and the reaction product (NH3 ) was determined by Nessler’s reagent. 3.8. Stability of the immobilized enzyme The stability of the soluble and the immobilized ErlASNase in 50 mM Tris–Cl buffer pH 8.2 was studied at 4 ◦ C. The rate of inactivation was measured by periodically removing samples for assay of enzymatic activity. Observed rates of inactivation (kin ) were deduced from plots of % of remaining activity versus time (days). Kinetic data of inactivation were analyzed as described in Section 4.5 using the computer program GraFit (Erithacus Software Ltd.) (Leatherbarrow, 1998). 3.9. N-terminal amino acid sequence analysis N-terminal amino acid sequence of the purified ErlASNase was carried out on a gas-phase Applied 542 Biosystems Protein sequencer, model 470A, equipped with an on-line phenylthiohydantoin analyzer, model 120 A. 3.10. Electrophoresis SDS polyacrylamide gel electrophoresis was performed according to the method of Laemmli (1970)

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Fig. 1. (A) Sequence alignments of type II l-ASNases from Erwinia species. The alignments were produced using ClustalW (Thompson et al., 1994). The secondary structure of l-ASNase from Er. chrysanthemi (PDB code 1hg0) and numbering are shown above the alignment. Alpha helices and beta strands are represented as helices and arrows, respectively, and beta turns are marked with TT. NCBI accession number for lASNases are in brackets: Seq1: Er. chrysanthemi 3937 (AAS67028); Seq2: Er. chrysanthemi NCPPB 1125 (CAA31239); Seq3: Er. chrysanthemi NCPPB 1066 (CAA32884); Seq4: Er. carotovora subsp. Atroseptica (AAS67027); Seq5: Er. carotovora (AAP92666). (B) Identity (%) between the available type II l-ASNase sequences from Erwinia species.

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on a slab gel containing 12.5% (w/v) polyacrylamide (running gel) and 2.5% (w/v) stacking gel. The protein bands were stained with Coomassie Brilliant Blue R-250. 3.11. Nucleotide sequence accession number The nucleotide sequence of Erl-ASNase has been submitted to NCBI and assigned the accession number: AY560098.

4. Results and discussion 4.1. Cloning, expression, and purification of Erl-ASNase The commercial availability of l-ASNases has revolutionized the molecular therapy of leukaemia. Thus, the characterization of new recombinant l-ASNases and the development of rapid, simple and effective production methods are not only of academic interest but also of practical importance (Avramis and Panosyan, 2005). The present work was undertaken in order to characterize a new recombinant l-ASNase from the pectobacterium Er. chrysanthemi 3937. The genome of this microorganism is currently being sequenced, and it is considered to be a model. A BLASTN search of the incomplete Er. chrysanthemi 3937 genome sequence (Sanger Centre database, http://www.sanger.ac.uk/) using the nucleotide sequence of l-ASNase from Er. chrysanthemi NCPPB 1125 (Filpula et al., 1988) as a query, yielded a high-probability match to a single 1047-bp open reading frame (ORF). This ORF encodes a predicted protein of 348 amino acids with 91% sequence identity with the query sequence. Fig. 1 shows amino acid sequence alignments resulting from the BLASTP search of the available l-ASNases from Erwinia species. It is interesting to note that an unexpectedly low sequence homology was observed

between different Erwinia species (e.g. 75–77% sequence identity was observed between Er. chrysanthemi and Er. carotovora enzymes), as well as between different subspecies (e.g. 91% sequence identity was observed between Er. chrysanthemi 3937 and Er. chrysanthemi NCPPB 1125). Bacterial l-ASNases are extracellular enzymes; thus, an Erl-ASNase precursor should possess a signal peptide sequence to direct its transport across the periplasmic membrane. Indeed, a putative signal peptide can be found at the N-terminal end, comprising residues 1–21. This sequence meets most of the criteria for a signal peptide as described by Perlman and Halvorson (1983). In order to determine whether the putative Er. chrysanthemi 3937 protein was indeed a functional l-ASNase, the full-length gene was cloned into the T7 expression vector pCR® T7/CT-TOPO. This plasmid was used to transform the expression host E. coli BL21(DE3)pLysS. Cell-free extracts of the E. coli transformants showed high asparaginase activity with a specific activity of 7.7 U/mg protein. Erl-ASNase was purified to homogeneity from E. coli cell-free extracts, in a single chromatographic step (15.4-fold purification with 69.8% yield) using cation exchange chromatography on S-Sepharose FF column. The theoretical isoelectric point of Erl-ASNase is 8.57; thus, at pH 5.5 the enzyme was quantitatively adsorbed to the cation exchanger whereas the endogenous E. coli l-ASNase (pI 4.7) was washed off. The results from a typical purification run are summarized in Table 1. The purity of the final Erl-ASNase preparation was evaluated by SDS-PAGE, which showed the presence of a single polypeptide chain (Fig. 2). Furthermore, the purity of the enzyme preparation was assessed by N-terminal amino acid sequence analysis. N-terminal amino acid sequencing gave the identity of a single pentapeptide matching the theoretical sequence of mature polypeptide (Ala-Asp-Lys-Leu-Pro) derived from the gene sequence (Fig. 1).

Table 1 Purification of recombinant l-ASNase from Erwinia chrysanthemi 3937 on S-Sepharose FF Step

Volume (ml)

Protein (mg)

Activity (Units)

Specific activity

Yield (%)

Purification (fold)

Crude extract S-Sepharose FF

10 8

3.3 0.15

25.5 17.8

7.7 118.7

100 69.8

1 15.4

Enzyme activity was measured at 37 ◦ C using Nessler’s reagent. Protein concentration was determined by the method of Bradford (1976).

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Fig. 2. SDS-polyacrylamide gel electrophoresis of Erl-ASNase purification on S-Sepharose FF cation exchanger. Protein bands were stained with Coomassie Brilliant Blue R-250. Left lane, E. coli crude extract after induction with 1 mM IPTG; middle lane, unbound protein fraction; right lane, Erl-ASNase eluted from S-Sepharose FF cation exchanger.

4.2. Kinetic analysis Molecular models of l-ASNase from Er. chrysanthemi 3937 were constructed to determine the location of residues that are not conserved between this enzyme and the enzyme derived from Er. chrysanthemi NCPPB 1125, the crystal structure of which has been determined by X-ray crystallography (Aghaiypour et al., 2001b). Comparison of the final models with the known crystal structures confirmed that the side-chain conformations of the Erl-ASNase active site residues are likely to be the same as those observed for the corresponding residues in the Er. chrysanthemi NCPPB 1125 enzyme. All differences in amino acid sequences

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are located away from the active site, and are thus not considered essential (Fig. 3A). The kinetic properties of the recombinant ErlASNase were investigated, and the kcat and Km parameters for a number of substrates were determined by steady-state kinetic analysis (Table 2). Comparison of the kinetic data presented in Table 2 allows analysis of the substrate binding site. Although the enzyme is capable of hydrolyzing a number of asparagine analogues, it is highly specific for l-Asn (Table 2). The Km values for l-Asn and l-Gln were determined to be 0.058 ± 0.013 and 6.7 ± 1.1 mM, respectively. This is probably due to several more favourable interactions with l-Asn compared to the other substrates, as has been demonstrated previously (Aghaiypour et al., 2001b). The enzyme showed a lower Km for l-Asn as compared to l-ASNases from Er. carotovora (Howard and Carpenter, 1972; Krasotkina et al., 2004; Kotzia and Labrou, 2005) whereas a higher Km value was found for l-Gln as compared to l-ASNases from Er. carotovora (Krasotkina et al., 2004), E. coli (Derst et al., 2000) and Er. chrysanthemi (Moola et al., 1994). For example, the Km (mM) for l-Gln is 3.4, 3.5 and 1.4 for enzymes from Er. carotovora, E. coli and Er. chrysanthemi (strain NCPPB 1125), respectively. Low affinity for l-Gln is very important, since the toxicity of the enzyme is partially attributable to the l-glutaminase activity (Howard and Carpenter, 1972). l-ASNases with high affinity for l-Asn and low affinity for l-Gln have been reported to be less troublesome during the course of anti-cancer therapy (Hawkins et al., 2004). The ␣-carboxyl group of the substrate appears to be an absolute requirement for catalysis. This requirement is demonstrated by the inability of the enzyme to hydrolyze ␤-alanine amide (descarboxyl asparagine), while succinamic acid (desaminoasparagine) is hydrolyzed. Analysis of structural mod-

Table 2 Kinetic parameters of ErASNase Substrates

Km (mM)

kcat (×103 s−1 )

kcat /Km (×103 mM−1 s−1 )

l-Asn l-Gln Succinamic acid Nα -Acetyl-l-Asn ␤-Alanine amide

0.058 ± 0.0131 6.7 ± 1.1 18.8 ± 4.8 0.80 ± 0.09 ND

23.8 ± 1.1 4.3 ± 0.5 23.9 ± 4.5 10.8 ± 0.2 ND

411.8 0.6 1.3 13.4 ND

ND: no detected. Steady-state kinetic measurements were performed at 37 ◦ C in 0.1 M Tris–HCl buffer, pH 8 (or pH 8.2 for l-Gln). All initial velocities were determined in triplicate. The kinetic parameters kcat and Km were calculated by non-linear regression analysis of experimental steady-state data using the GraFit (Erithacus Software Ltd.) program.

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Fig. 3. Structural representations of the Er. chrysanthemi 3937. (A) Diagram of the modelled enzyme subunit with l-Glu bound to the active site. The bound ligand is shown in a stick representation. Different residues between Er. chrysanthemi 3937 and Er. chrysanthemi NCPPB 1125 are shown in space fill representation. (B) Structural representation of the active sites of two modelled complexes of Er. chrysanthemi 3937. (I) with succinic acid and (II) with l-Glu. Bound substrates are shown in a stick representation and drawn in yellow. Hydrogen bonds are shown as dotted lines. The figure was created by PyMOL (DeLano, 2002). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

els in complex with l-Glu and succinic acid (Fig. 3B) showed that the ␣-carboxyl group of the ligands interacts with the enzyme in a conserved manner. One of the oxygen atoms interacts with the amide group of Asp117 and with the ␥-OH group of Ser83. The other oxygen atom forms a hydrogen bond with the amide group of Ser83.

The ␣-amino group of the substrate is also important but not essential for catalysis, as indicated by the ability of the enzyme to hydrolyze N␣ -acetyl-l-Asn and succinamic acid at a lower efficiency (kcat /Km ) compared to that for l-Asn. It has been suggested that the ␣amino group contributes to the proper orientation of the substrate and influences the rate of catalysis, substrate

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affinity, and substrate specificity (Aghaiypour et al., 2001b; Kotzia and Labrou, 2005). The ␣-amino group of the ligands participates in two conserved hydrogen bonds formed with the carboxyl oxygen atoms of Asp117 and Glu84 (Fig. 3B). In addition, the distance between the carboxyl group and the amide group on the substrate appears to contribute to the rate of catalysis and the Km value. The 4-carbon dicarboxylic acid monoamides (l-Asn, Nα -acetyl-l-Asn and succinamic acid) have higher kcat values than the 5-carbon dicarboxylic acid monoamide l-Gln. Considering the modelled active site structure of the enzyme, the ␥carboxyl group of succinic acid forms two pairs of hydrogen bonds, one pair with the main-chain nitrogen atoms of Thr36 and Thr118, and another pair with the ␥-OH group of Thr118 and the main-chain oxygen of Ala141. The larger side chain of l-Glu forms two hydrogen bonds with Thr118, one with its ␥-OH group and the second with the main-chain nitrogen atom. In addition, the orientation of the ␦-carboxyl group of l-Glu in the active site is different than observed for succinic acid (Fig. 3B). This orientation has been suggested to influence the conformation of the active site flexible loop (residues 20–60 approx.), which was found to be disordered in the complex of Er. chrysanthemi l-ASNase with l-Glu (Aghaiypour et al., 2001b). Thus, it is conceivable that both fewer bonds and conformational changes have important roles in the lower activity of Erl-ASNase for l-Gln than for l-Asn. 4.3. Effect of temperature on the hydrolysis reaction To gain a deeper understanding of the mechanism of catalysis by Erl-ASNase, the temperature dependence of its catalytic activity was investigated. The effect of temperature on l-Asn hydrolysis was determined. The activity data were analyzed by plotting ln Vmax against 1/T and fitting them to the Arrhenius equation (Eq. (1)) (Peng et al., 2003): ln Vmax = ln Z −

Ea RT

(1)

where Ea is the energy of activation, R the gas constant, and Z is the pre-exponential factor. The activity of ErlASNase increased with increasing temperature from 5 to 35 ◦ C (Fig. 4). Fitting of the experimental data to Eq. (1) showed a linear Arrhenius plot, and activation

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Fig. 4. Effect of temperature on Erl-ASNase activity. The activity data were fitted to the Arrhenius equation. The line represents the best least-square fit of the activity data. Erl-ASNase activity was measured between 5 and 35 ◦ C ().

energy that is well within the range of other enzymecatalyzed reactions (Table 3, Fan et al., 2000; Peng et al., 2003; Kotzia and Labrou, 2005). According to Eyring’s transition state theory, the temperature dependence of kcat is given by Eq. (2) (Fan et al., 2000): kcat =

k␤ T −(G= /RT ) k␤ T −(H = /RT −S = /R) = e e h h (2)

where k␤ is the Boltzmann constant, h the Planck’s constant, R the gas constant, and G= , H= and S= are the free energy, enthalpy, and entropy of activation of the rate-limiting step of the reaction, respectively. The data are plotted as ln(kcat /T) against 1/T, and fitted to Eq. (2). The results are shown in Table 3. A positive H= was expected because the transition state involves the breaking of bonds. The S= calculated was negative, indicating that the formation of the transition state requires the reacting molecules to be more constrained in the activated complex, and that the rate-limiting step possibly results from less tight hydrophobic interactions. Table 3 Thermodynamic parameters of the l-Asn hydrolysis reaction of ErlASNase Temperature range (◦ C) Ea (kJ/mol) H= (kJ/mol) S= (J/mol K)

5–35 20.7 18.3 −157.03

Data were analyzed by Arrhenius (Eq. (1)) and Eyring (Eq. (2)) plots.

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Fig. 5. Thermal inactivation curves of Erl-ASNase at pH 5.5 (䊉), pH 7 () and pH 8.5 (). The residual activities of the Erl-ASNase were measured after heat treatment at various temperatures for 7.5 min. Experiments were performed in triplicate and the standard deviation was less than 5%.

4.4. Thermal stability of Erl-ASNase To investigate the structural stability of ErlASNase, heat inactivation studies were carried out at pH 5.5, 7 and 8.5 (Eijsink et al., 2005). Fig. 5 shows the inactivation process of Erl-ASNase at these three pH values. The enzyme showed higher stability at acidic to neutral pH values, whereas lower stability was observed at alkaline pH. The inactivation profiles at pH 5.5 and 7 were biphasic and showed two clear transitions with inflection points corresponding to Tm values of 46.6 and 62.5 ◦ C at pH 5.5, and 46.4 and 57.2 ◦ C at pH 7, respectively. The biphasic profiles indicated

the presence of intermediate active species along the unfolding pathway of Erl-ASNase (Sturtevant, 1987; Freire et al., 1990). On the other hand, at pH 8.5, a single transition corresponding to a Tm value of 42.5 ◦ C was observed. The lower stability found at alkaline pH may be related to the fact that the pH 8.5 is very close to the isolectric point of the enzyme (theoretical pI 8.57). The time course of thermal inactivation of ErlASNase was investigated at temperatures of therapeutic importance (37 and 41 ◦ C). The results indicate that the enzyme is particularly stable at 37 ◦ C. On the other hand, at 41 ◦ C the enzyme shows significantly lower thermostability. Kinetic analysis of the thermal inactivation at 37 and 41 ◦ C showed linear plots (Fig. 6) from which the first-order inactivation rate constants kins were calculated according to Eq. (3): log(% remaining activity) = 2.303kin t

(3)

At 37 ◦ C, the enzyme exhibits a kin of 1.7 × 10−3 h−1 , whereas at 41 ◦ C kin = 48 × 10−3 h−1 , which is about 28-fold higher than that at 37 ◦ C. Comparing the thermostability of Erl-ASNase with that of l-ASNase from E. coli, the latter shows a half-life of 4.6 h at 37 ◦ C (Zhang et al., 2004). 4.5. Activity and stability of immobilized Erl-ASNase on epoxy-activated sepharose The immobilization of enzymes has proven to be very useful in biotechnology, as it usually leads to

Fig. 6. Time course of inactivation of Erl-ASNase at 37 ◦ C (left) and 41 ◦ C (right). The rate of inactivation was measured by periodically removing samples for assay of enzymatic activity. The lines were generated by least-square fitting of the experimental data points.

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enhanced operational and storage stability (KatchalskiKatzir et al., 2000; Hibbert et al., 2005). Therapeutic application of bacterial l-ASNases leads to an immune response that can stimulate production of IgE or other immunoglobulins. Anaphylactic reactions are observed in about 33% of patients (Kravtzoff et al., 1996; Muller et al., 2001). Thus, numerous modifications and coupling studies have been carried out to improve pharmacodynamic parameters and efficacy, and to minimize the immunogenic effects seen in the clinical use of l-ASNase (Jorge et al., 1994; JeanFranc¨eois et al., 1997; DeLoach et al., 1990). In this paper, we report that the Erl-ASNase can be efficiently immobilized on epoxy-activated Sepharose CL-6B using 1,4-butanediol diglycidyl ether as a coupling agent. Immobilization was carried out in the presence or absence of succinic acid. The immobilized enzyme showed about 60% of the activity of the native enzyme, indicating that the method of immobilization is particularly suitable for Erl-ASNase. The hydrolysis of l-Asn by immobilized ErlASNase was chosen as a model reaction. The turnover of 8.5 mM l-Asn by immobilized Erl-ASNase is illustrated in Fig. 7. The presence of succinic acid in the immobilization reaction had no significant effect on the turnover of the enzyme, as shown in Fig. 7A. In addition, the stability of the immobilized Erl-ASNase was also investigated at 4 ◦ C and pH 8.2, and the results are shown in Fig. 7B. When the immobilized ErlASNase was incubated at pH 8.2 and 4 ◦ C, the enzyme was progressively inactivated. The kinetics of inactivation were biphasic, with a rapid inactivation occurring immediately (up to 48 h), to yield enzyme of about 43% residual activity, and a slow inactivation continuing for more than 60 days. Rate constants for the biphasic inactivation reaction were calculated using Eq. (4) (Bailey and Colman, 1985; Kotzia and Labrou, 2004): remaining activity = (1 − F )e−kfast t + F e−kslow t

(4)

where F represents the fractional residual activity of the partial active enzyme intermediate and kfast and kslow are the rate constants for the slow and fast phases of the reaction, respectively. The immobilized enzyme shows inactivation rates of 0.702 ± 0.16 and 3.3 × 10−3 ± 4 × 10−4 d−1 for the fast and slow phases of inactivation, respectively. The enzyme immobilized in the presence of succinic acid exhibits inactiva-

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Fig. 7. (A) Turnover of 8.5 mM l-Asn by the free () and immobilized Erl-ASNase. The enzyme was immobilized in the presence (䊉) or in the absence () of Suc. Turnover was monitored by determining the amount of released ammonia. The curves were generated by least-squares fitting of the experimental data points. (B) Stability of free () and immobilized Erl-ASNase at 4 ◦ C and pH 8.2. The enzyme was immobilized in the presence (䊉) or in the absence () of Suc.

tion rates of 0.672 ± 0.05 and 3.6 ± 2.6 × 10−4 d−1 . On the other hand, the free enzyme shows significantly lower stability and loses its activity within 15 days. These results demonstrate that the immobilized enzyme shows high stability; thus, the approach offers a new method of designing an Erl-ASNase bioreactor for depletion of the circulating pools of l-Asn that can be operated over a long period of time with high efficiency. In conclusion, in the present study a new l-ASNase from Er. chrysanthemi 3937 has been cloned, expressed and characterized. The results of the present work

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