Engineering thermal stability of L-asparaginase by in vitro directed evolution Georgia A. Kotzia and Nikolaos E. Labrou Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Greece Bacterial l-asparaginases (l-ASNases, EC 3.5.1.1) have been used as therapeutic agents in the treatment of lymphoblastic leukaemia [1,2]. l-ASNase exerts its antitumor activity by depleting the nonessential amino acid l-Asn from human blood and other extracellular fluids [3,4]. Certain malignant cells, unlike normal cells, are unable to synthesize l-Asn due to the lack of l-asparagine synthetase activity [5,6]. These cells are dependent on extracellular sources of l-Asn in order to complete protein synthe- sis [1,7]. Therefore, administration of l-ASNase selec- tively destroys the neoplastic cells by starving them of l-Asn [8,9]. To date, l-ASNases of Erwinia chrysanthemi and Escherichia coli are in clinical use, as effective drugs in the treatment of acute lymphoblastic leukaemia, Hodg- kin’s disease, acute myelocytic leukaemia, acute myelo- monocytic leukemia, lymphosarcoma, melanosarcoma, etc. [10,11]. The main restrictions to the therapeutic use of l-ASNase include its premature inactivation, thus necessitating frequent injections to maintain ther- apeutic levels, and several types of side reactions from mild allergies and the development of immune responses to dangerous anaphylactic shock [9,12–14]. Over the years, many homologous l-ASNases have been cloned and characterized to find enzymes with Keywords directed evolution; enzyme engineering; leukaemia; saturation mutagenesis; thermal stability Correspondence N. E. Labrou, Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece Fax: +30 210 5294308 Tel: +30 210 5294308 E-mail: lambrou@aua.gr (Received 19 September 2008, revised 2 December 2008, accepted 19 January 2009) doi:10.1111/j.1742-4658.2009.06910.x l-Asparaginase (EC 3.5.1.1, l-ASNase) catalyses the hydrolysis of l-Asn, producing l-Asp and ammonia. This enzyme is an anti-neoplastic agent; it is used extensively in the chemotherapy of acute lymphoblastic leukaemia. In this study, we describe the use of in vitro directed evolution to create a new enzyme variant with improved thermal stability. A library of enzyme variants was created by a staggered extension process using the genes that code for the l-ASNases from Erwinia chrysanthemi and Erwinia carotovora. The amino acid sequences of the parental l-ASNases show 77% identity, but their half-inactivation temperature (T m ) differs by 10 °C. A thermo- stable variant of the E. chrysamthemi enzyme was identified that contained a single point mutation (Asp133Val). The T m of this variant was 55.8 °C, whereas the wild-type enzyme has a T m of 46.4 °C. At 50 °C, the half-life values for the wild-type and mutant enzymes were 2.7 and 159.7 h, respec- tively. Analysis of the electrostatic potential of the wild-type enzyme showed that Asp133 is located at a neutral region on the enzyme surface and makes a significant and unfavourable electrostatic contribution to overall stability. Site-saturation mutagenesis at position 133 was used to further analyse the contribution of this position on thermostability. Screen- ing of a library of random Asp133 mutants confirmed that this position is indeed involved in thermostability and showed that the Asp133Leu muta- tion confers optimal thermostability. Abbreviations Eca L-ASNase, L-asparaginase from Erwinia carotovora; ErL-ASNase, L-asparaginase from Erwinia chrysanthemi 3937; L-ASNase, L-asparaginase; StEP, staggered extension process; T m, half-inactivation temperature. 1750 FEBS Journal 276 (2009) 1750–1761 ª 2009 The Authors Journal compilation ª 2009 FEBS less toxic effects [15,16]. In addition, many attempts have been made with a view to extending the half life of l-ASNase in the blood circulation [17,18], with the most promising approach being its covalent coupling to poly(ethylene glycol) [19–21]. Unfortunately, naturally available enzymes are usu- ally not optimally suited for therapeutic purposes. This incompatibility often relates to the stability of the enzymes under body conditions. In the case of engineering proteins for thermostability, researchers are in the enviable position of being able to choose between three different, apparently equally successful, strategies: rational design, directed evolution and the construction of (semirational) synthetic consensus genes. Although there are many examples of enzymes that have been stabilized by the introduction of only one or two mutations [22] and despite many success- ful efforts to understand the structural basis of pro- tein stability, there is still no universal strategy to stabilize ‘any’ protein by a limited number of ratio- nally designed mutations. Well-known and reasonably successful types of rational engineering work inc- lude rigidifying mutations (e.g. Xxx fi Pro or Gly fi Xxx or the introduction of disulfides), work- ing primarily through their effect on the entropy of the unfolded state, improvement of molecular packing (e.g. shortening of loops, improvement of interactions in the hydrophobic core, for example, by the removal of internal cavities), modification of surface charge networks or reinforcement of a higher oligomerization state [23]. Because the structure–function relationship is not known or fully understood for the majority of proteins, directed evolution provides a powerful approach for improving thermostability [24–27]. This method, in combination with high-throughput screen- ing, can be used even in cases where no information on the 3D structure exists, and a single experiment may provide enough variants to obtain the best thermostable mutant [28]. In this study, l-ASNases from Erwinia carotovora (Ecal-ASNase) [15] and Erwinia chrysanthemi (Erl- ASNase) [16] were subjected to directed evolution, with a view to find variants with improved thermosta- bility. Enhancing the stability of l-ASNase by protein engineering improves its body-residence time, and thereby minimizes immunosuppressive effects by lower- ing the therapeutic dose. After one round of directed evolution one variant with a point mutation (Asp133Val) was found. An extensive search for the best-fit residue at position 133 was made using satura- tion mutagenesis, which revealed that this site plays an important role in the thermal stability of the enzyme. The present work represents the first experimental approach for improving the thermal stability of this therapeutic important enzyme. Results and Discussion Identification and kinetic characterization of a thermostable mutant One of the most important goals of protein engineer- ing is to produce modified enzymes with improved thermostability. However, the thermal stability of a protein is not readily predictable from its 3D structure. Thus, directed evolution is by far the best way to alter this enzyme property [24–28]. In this study, we used in vitro directed evolution using two homologous l-ASNase sequences from E. carotovora and E. chrysanthemi (77% sequence identity). The library of mutated genes was generated using the staggered extension process (StEP) and expressed in E. coli. Wild-type and mutant enzymes were expressed as non-tagged proteins. The diversity of the DNAs in the resulting library was examined by sequence analysis of 10 randomly picked clones, and the results showed a satisfactory variability in recombi- nations. The thermal stability of the enzyme variants was evaluated at 55 °C. Under these conditions, wild- type enzymes from E. carotovora and E. chrysanthemi displayed 19.9% and 37.2% remaining activity, respec- tively. One clone that showed 87.3% remaining activity was identified and selected for further study. This enzyme variant was sequenced and found to be a single point mutant of the E. chrysanthemi enzyme. The mutation was at position 133 of the amino acid sequence, with Asp replaced by Val (codon GAC fi GTC). Shis mutation was the result of an error introduced by the DNA polymerase. Following sequencing, the mutant enzyme was sub- jected to purification according to a purification proce- dure established for the wild-type enzyme using an S-Sepharose FF column [16]. The results showed that the mutant enzyme hardly bound to the cation exchan- ger. For example, the dynamic capacities for the wild- type and mutant enzymes were determined to be 8.9 UÆmL )1 adsorbent and 0.1 UÆmL )1 adsorbent, respectively. Thus, a new purification protocol was developed using a two-step chromatographic procedure involving a negative purification step using DEAE– Sepharose CL6B followed by immobilized metal chelate-affinity chromatography on a Ni-NTA column (Fig. 1). It is of particular importance to point out that the Asp133Val mutant enzyme showed unusual chromatographic behaviour on ion exchangers. In particular, at pH 5.5, the enzyme did not bind to the G. A. Kotzia and N. E. Labrou Engineering thermal stability of L-asparaginase FEBS Journal 276 (2009) 1750–1761 ª 2009 The Authors Journal compilation ª 2009 FEBS 1751 cation exchanger (S-Sepharose FF column) or to the anion exchanger (DEAE–Sepharose CL6B), although its theoretical isoelectric point is 8.57. This was the first indication that the mutant enzyme exhibits unu- sual electrostatic potential. The basis of thermal stability of the Asp133Val mutant The thermal stability of the purified Asp133Val mutant enzyme was assessed by measuring its residual activity after heat treatment for 7.5 min at various tempera- tures (Fig. 2). The half-inactivation temperature (T m ) of the mutant enzyme was found to be 55.8 °C, which is almost 9.4 °C higher than that of the wild-type Erl- ASNase [16]. Kinetic analysis of the thermal inactiva- tion of the wild-type and Asp133Val mutant enzymes at 50 °C gave linear plots (Fig. 3). For both enzymes, the inactivation process followed first-order kinetics [29]. The inactivation rate constant was calculated using the following equation: log(% remaining activity) = 2.303 Á k in Á t where k in is the inactivation constant. k in for the Asp133Val mutant enzyme was found to be 4.34 · 10 )3 h )1 , which is 59-fold lower than that of wild-type Erl-ASNase. The half-lives (t 1 ⁄ 2 )at50°Cof the wild-type and mutant enzymes were determined to be 2.7 and 159.7 h, respectively. To gain a deeper insight into the structural basis of thermal stability, a molecular model of the mutant was constructed (Fig. 4). Analysis of the structure showed that Asp133 lies at the external surface of Erl-ASNase, in particular at a loop region formed by residues Thr129–Lys134 between a4 and b4 (Fig. 4A). It forms a salt bridge and two hydrogen bonds with Arg169. The first hydrogen bond is formed between the OD1 atom of Asp133 (2.87 A ˚ ) and the side chain NH 2 of Arg169, whereas the OD2 atom forms a weak H-bond (3.47 A ˚ ) with the side chain NH 2 of Arg169 (Fig. 4B). In the Asp133Val model, these two hydrogen bonds are lost and the side chain of Val133 no longer inter- acts with Arg169 (Fig. 4C). Fig. 2. Thermal inactivation curves. The residual activities of the wild-type (h) and mutant Asp133Val ( ) enzymes were measured after heat treatment at various temperatures (30–70 °C) for 7.5 min. Fig. 1. SDS ⁄ PAGE of Asp133Val mutant enzyme purification. Pro- tein bands were stained with Coomassie Brilliant Blue R-250. Lane A, molecular mass markers; lane B, E. coli BL21 (DE3)pLysS crude extract after induction with 1 m M isopropyl thio-b-D-galactoside; lane C, unbound fraction from DEAE-Sepharose CL6B, pH 7.5; lane D, eluted Asp133Val mutant enzyme from the Ni-NTA affinity adsorbent. Fig. 3. Kinetics of thermal inactivation of the wild-type and Asp133Val mutant enzyme. The residual activities of the wild-type ( ) and mutant Asp133Val (¤) were measured at various times after incubation at 50 °C. The results are presented as plot of log(% remaining activity) versus time (h). Engineering thermal stability of L-asparaginase G. A. Kotzia and N. E. Labrou 1752 FEBS Journal 276 (2009) 1750–1761 ª 2009 The Authors Journal compilation ª 2009 FEBS Thus, any attempt to understand the greater thermal stability of the Asp133Val mutant enzyme in terms of intramolecular interactions (e.g. H-bonding, ionic bonds) in the structure was unsuccessful. Instead, the explanation appears to lie in the much greater entropy of activation in the case of the wild-type enzyme. In principle, the entropy of inactivation consists of two parts: the increased configurational entropy due to partial unfolding of the protein in the transition state and the decrease in entropy of the solvent due to expo- sure of hydrophobic side chains. The latter effect is likely to be small at elevated temperatures [30] and the major contribution must then be the increase in config- urational entropy. Thus, the greater rate of thermal inactivation of the wild-type enzyme than of the mutant is probably mainly due to greater flexibility or disordering of the transition state with some contribu- tion from a lower degree of exposure of hydrophobic residues. To assess whether Asp133 contributes to structural flexibility, we analysed the plots of the crys- tallographic B-factors along the polypeptide chain of the enzyme structure. This plot can give an indica- tion of the relative flexibility of portions of the protein [15,31]. As shown in Fig. 5, the structure dis- plays a well-defined flexibility pattern. Several highly mobile regions throughout the entire sequence can be identified, and these are separated by a number of segments with low mobility. Asp133 displays high Fig. 4. Structural representations of the wild-type and Asp133Val mutant enzyme. (A) Diagram of the modelled Asp133Val mutant enzyme subunit with succinamic acid bound to the active site. The bound ligand and the mutated residue (Val133) are shown in a stick representation and are labelled. (B) Structural representation of the mutation site 133 of the wild-type ErL -ASN- ase. (C) Structural representation of the mutation site 133 of the Asp133Val mutant. Hydrogen bonds are shown as dashed lines and residues are labelled. The model of the mutated enzyme was constructed using WHAT IF [51]. (D) A closer view of the electro- static potential of the mutation site 133. Negative, positive and neutral values of electrostatic potential are indicated by shades of red, blue and white colour, respectively. (E) Superposition of the Pois- son–Boltzmann electrostatic potential of the mutant and the wild-type enzymes. Asp113 is shown as a spacefill representation (col- oured black) and labelled. The colour code utilized to represent the electrostatic poten- tial along with the potential range is: red: )1.8; white: 0; blue: 1.8. All figures were created using PYMOL [54], except (E) which was created using SWISS-PDB VIEWER [53]. G. A. Kotzia and N. E. Labrou Engineering thermal stability of L-asparaginase FEBS Journal 276 (2009) 1750–1761 ª 2009 The Authors Journal compilation ª 2009 FEBS 1753 crystallographic B-factors located at a region with high mobility, indicating that this residue undergoes large fluctuations. This may cause local disordering with increased flexibility, which may contribute to the increase in configurational entropy. Erl-ASNase is composed of four identical subunits, and the active enzyme is always a tetramer. To deter- mine whether the higher thermal stability observed for the Asp133Val mutant enzyme is due to higher stabil- ity of the quaternary or tertiary structure, subunit- dissociation experiments were carried out. Shifrin et al. [32] showed that guanidinium chloride dissociates the enzyme tetramer in 50 mm phosphate buffer at pH 7.5. The dissociation is accompanied by the appearance of an ultraviolet difference spectrum with a maximum at 288 nm. The band at 288 nm in the difference spec- trum has been ascribed to tyrosyl residues [32]. In this study, the rate of subunit dissociation in the wild-type and Asp133Val mutant enzymes was monitored by following the rate of appearance of the 288 nm band in the ultraviolet difference spectrum. As illustrated in Fig. 6, the rate of dissociation of the tetramer was found to be approximately equal for both enzymes (first-order rate constants: 0.011 and 0.013 min )1 for the wild-type and Asp133Val mutant enzymes, respec- tively), indicating that the stabilizing effect of the mutation is not due to quaternary structure stabiliza- tion. Instead, it is the result of stabilization of the tertiary structure. Taking into account the unusual chromatographic behaviour of the mutant enzyme on ion exchangers, and because in silico structural analysis of the interac- tions in the microenvironment of Val133 in the mutant enzyme did not provide adequate explanations for its higher thermostability, electrostatic potential analysis was performed. Analysis of the wild-type enzyme showed that Asp133 is located at an uncharged (neu- tral) region of the enzyme (Fig. 4D). Replacement of a surface charge (Asp) with a hydrophobic residue (Val) would not normally be expected to increase the stabil- ity of the protein, because for a hydrophobic side chain it is more favourable to be buried within the core of the protein than to be exposed to solvent. It has been suggested, however, that in some cases there can be residues located on the surface of a protein that provide an unfavourable electrostatic contribution to the overall stability of the domain, due to the asymme- try of the electrostatic potential mapped on the surface of the protein [33]. In our case, because Asp133 lies in a neutral environment, its charge is unfavourable and it may therefore destabilize the overall structure. Com- parison of the Poisson–Boltzmann electrostatic poten- tial of the mutant and the wild-type (Fig. 4E) showed that greater symmetric positive potential was observed in the mutant enzyme than in the wild-type enzyme. This may cause less structural perturbations in the mutant enzyme. Effects of the Asp133Val mutation on kinetic parameters The mutant enzyme was subjected to steady-state kinetic analysis using three different substrates: l-Asn, l-Gln and N a -acetyl-Asn (Table 1). The results showed that the k cat and K m values for l-Asn were increased by 1.6- and 2.9-fold, respectively, compared with the wild-type enzyme. By contrast, the K m values for the Fig. 5. The dynamics of ASNase. A plot of the crystallographic B-factors along the polypeptide chain obtained from the crystal structure of E. chrysanthemi ASNase, (PDB code 1O7J). The plot was produced using WHAT IF software package [51]. The height at each residue position indicates the average B-factor of all atoms in the residue. B-factors are available in the PDB file 1O7J. Fig. 6. Guanidinium chloride induced subunit dissociation. Rate of appearance of the 288 nm band of the wild-type (s) and mutant Asp133Val (d), as a function of time in 0.05 M phosphate buffer, pH 7.5, containing 3 M guanidine chloride. Changes in absorbance at 288 nm were monitored for 5 min. Engineering thermal stability of L-asparaginase G. A. Kotzia and N. E. Labrou 1754 FEBS Journal 276 (2009) 1750–1761 ª 2009 The Authors Journal compilation ª 2009 FEBS l-Gln and N a -acetyl-l-Asn were reduced and k cat values were increased (Table 1). The increased K m value for l-Asn, although undesirable, is still within the range of acceptability for therapeutic applications [35]. The results of the kinetic analysis indicate that although the site of the mutation is distant from the active site, it makes a significant contribution to catalysis (k cat and K m ), suggesting that long-range effects play an important role. Considering the inter- action of the enzyme with the substrate, it is reason- able to propose that there may be a long-range effect in the active site. In particular, the mutation at posi- tion 133 may destabilize the crucial hydrogen bond formed between the side chain of the substrate and the main chain oxygen of Ala139. This interaction has been shown to be responsible for the higher mobility of the active-site loop and of the flexible cat- alytic region 120–140, and it is believed to contribute to substrate specificity and to the rate-limiting step [15,16,36]. These structural observations are consistent with the results of the kinetic analysis (Table 1) and presumably explain the effect of the mutation on kinetic constants. Similar long-range effects have been found in the serine protease subtilisin BPN’ [37]. For example, charged residues on the surface of the enzyme some 13–15 A ˚ from the active site have been found to modulate enzyme–substrate complex forma- tion and catalysis. Also, long-range interactions have been found in the case of aminoacyl-tRNA synthetase and 4-chlorobenzoyl-CoA dehalogenase catalysis [38,39]. Site-saturation mutagenesis at position 133 In vitro site-directed evolution (saturation mutagenesis) can be used to advantage during protein engineering to explore additional evolution pathways and enable rapid diversification in protein traits [40]. This method makes possible the creation of a library of mutants containing all possible mutations at one or more pre- determined target positions, in order to determine the best-fit residue at that position. It has been used suc- cessfully for rapid improvement of various protein functions [40,41]. In this study, site-saturation mutagenesis at posi- tion 133 was used to investigate in greater depth the contribution of this position to the thermostability. A library of enzyme variants was created by overlap extension PCR using two degenerate synthetic oligo- nucleotides in which the mutation site (position 133) was diversified using a randomized NNN codon. The library was subsequently screened for clones with improved stability at 60 °C for 5 min. Under these conditions, the mutant Asp133Val shows 21.3% resid- ual activity. Four clones that showed ‡ 45% residual activity (compared to the Asp133Val mutant) were selected and sequenced. Three clones were found to have single point mutations at position 133: Asp133Leu, Asp133Ile and Asp133Thr. In addition, Table 1. Kinetic parameters of the wild-type and mutant enzymes. Steady-state kinetic measurements were performed at 37 °C in 0.1 M Tris ⁄ HCl, pH 8 (or pH 8.2 for L-Gln). All initial velocities were determined in triplicate. The kinetic parameters k cat and K m were calculated by nonlinear regression analysis of experimental steady-state data using the GRAFIT (Erythacus Software Ltd, Staines, UK) program [34]. Enzymes Substrates K m (mM) k cat (s )1 )(· 10 3 ) k cat ÆK m )1 (mM )1 Æs )1 ) (· 10 3 ) Wild-type a L-Asn 0.058 ± 0.013 23.8 ± 1.1 411.8 L-Gln 6.7 ± 1.1 4.3 ± 0.5 0.6 N a -Acetyl-L-Asn 0.80 ± 0.09 10.8 ± 0.2 13.4 L-Asn 0.153 ± 0.021 37.94 ± 1.83 247.7 Asp133Val L-Gln 1.999 ± 0.236 16.03 ± 0.69 8.018 N a -Acetyl-L-Asn 0.597 ± 0.096 22.71 ± 0.66 38.04 L-Asn 0.160 ± 0.051 4.708 ± 0.64 29.425 Asp133Leu L-Gln 1.677 ± 0.278 0.815 ± 0.05 0.486 N a -Acetyl-L-Asn 1.788 ± 0.322 3.103 ± 0.21 1.735 L-Asn 0.097 ± 0.022 2.467 ± 0.19 25.433 Asp133Ile L-Gln 1.099 ± 0.094 0.677 ± 0.02 0.616 N a -Acetyl-L-Asn 5.613 ± 1.426 2.576 ± 0.35 0.459 L-Asn 0.038 ± 0.004 1.686 ± 0.03 44.368 Asp133Thr L-Gln 1.008 ± 0.061 0.646 ± 0.01 0.641 N a -Acetyl-L-Asn 2.096 ± 0.293 2.139 ± 0.12 1.021 a Data for the wild-type enzyme were from Labrou & Kotzia [16] and are included for comparison. G. A. Kotzia and N. E. Labrou Engineering thermal stability of L-asparaginase FEBS Journal 276 (2009) 1750–1761 ª 2009 The Authors Journal compilation ª 2009 FEBS 1755 one clone had a double mutation: Asp133Leu ⁄ Asp103Thr. The spontaneous Ala103Thr mutation (codon GCG fi ACG) was due to random error introduced by Pfu DNA polymerase. The four most thermostable clones have an uncharged substitution at position 133 (Leu, Ile, Thr). This further supports the finding that position 133 contributes significantly to the electrostatic potential of the enzyme, and it pro- vides evidence for the necessity of uncharged ⁄ hydro- phobic residue at position 133 for attainment high thermostability. The selected saturation variants were purified using the same procedure that was used for the Asp133Val mutant. Subsequently, their thermal stabilities were evaluated using heat-inactivation studies at 57.5 °C (Fig. 7) and the results are listed in Table 2. All mutants showed reduced k in values (from 1.75- to 3.2-fold) compared with the Asp133Val mutant, indi- cating further improvement in their thermal stability. The Asp133Leu mutant appeared to be the most thermostable, with a k in of 0.040 min )1 . The double mutant Asp133Leu ⁄ Ala103Thr showed a k in compa- rable with that of the single point mutant Asp133Leu. Structural analysis of 3D models of the mutant enzymes showed the formation of additional non- covalent interactions in the mutated enzymes (Fig. 8). In particular, the side chain hydroxyl group of the Asp133Thr mutant is involved in an H-bond with the side chain of Arg169, similarly to the wild- type Erl-ASNase (Fig. 4C). By contrast, the Asp133Leu and Asp133Ile mutants appear to form additional van der Waals interactions. The side chain of Leu133 interacts (van der Waals contacts) with the side chain of Asn226, and Ile133 interacts with Arg169 and Gly170. All residues are parts of loops; therefore, the additional stabilizing effects of the mutations, compared with the Asp133Val enzyme, may be due either to restriction of the conforma- tional freedom of the protein or to the energetic contribution of the newly formed H-bond and van der Waals contacts. Variants bearing a single mutation (Asp133Leu, Asp133Ile, Asp133Thr) were subjected to kinetic analysis using l-Asn, l-Gln and N a -acetyl-l-Asn as substrates. The results showed little to moderate effect of the Leu, Ile and Thr substitutions on the K m values compared with the Asp133Val mutant (Table 1). It is interesting to note that for the Asp133Thr mutant, the K m value for l-Asn was reduced fourfold compared with the Asp133Val mutant and by 1.5-fold compared with the wild-type Erl-ASNase. By contrast, the k cat values were significantly reduced although the enhanced specificity (k cat ÆK m )1 ) towards l-Asn was maintained in all mutants. Conclusions The results of this study provide new data on the structural basis of the thermal stability of E. chry- santhemi l-ASNase, and provide a basis for the design of new, improved forms of the enzyme for future ther- apeutic use. It has been shown that the hydrophobic effect, hydrogen bonding and packing interactions between residues in the interior of the protein are dominant factors that define protein stability. The role of surface residues in protein stability has received much less attention. The stability of the Asp113Val mutant enzyme is a particularly interesting rare case in which replacement of a surface charge with a hydrophobic residue leads to an increase in the stabil- ity of the protein. Whereas conventional chemical intuition would expect that salt bridges should contribute favourably to protein stability, recent Fig. 7. Kinetics of thermal inactivation of the mutant Asp133Val and its saturation variants. The residual activities of the mutant Asp133Val (e) and of the saturation variants were measured at var- ious times after incubation at 57.5 °C. The results are presented as plot of log(% remaining activity) versus time (min). Asp133Leu ( ), Asp133Ile (d) and Asp133Thr (D). Table 2. First-order inactivation rate constants (k in , min )1 ) of the mutant enzymes at 57.5 °C. Enzymes Rate constants k in (min )1 ) Asp133Val 0.123 ± 0.0053 Asp133Leu 0.043 ± 0.0004 Asp133Ile 0.071 ± 0.0015 Asp133Thr 0.055 ± 0.0007 Engineering thermal stability of L-asparaginase G. A. Kotzia and N. E. Labrou 1756 FEBS Journal 276 (2009) 1750–1761 ª 2009 The Authors Journal compilation ª 2009 FEBS computational and experimental evidence has shown that salt bridges can be either stabilizing or destabiliz- ing [41]. For example, alleviation of unfavourable sur- face charge can increase the stability of proteins [42]. Many proteins contain clusters of positively or nega- tively charged residues, and optimization of the surface electrostatic potential may enhance protein stability. For example, in recent studies of ribonucle- ase T1 and ubiquitin, it was shown that relieving surface charge through mutation increased protein stability [42–44]. During the development of a therapeutic protein, it is important to improve its long-term stability. Under- standing the stability parameters and the factors that affect them are critical steps. The thermostable enzyme variants reported in here may be tested further on ani- mals and ⁄ or humans in order to create a new drug for future therapeutic use. Experimental procedures l-Asn and l-Gln were obtained from Serva (Heidelberg, Germany). a-Ketoglutaric acid and Sepharose CL6B from Sigma (St Louis, MO, USA). N a -Acetyl-l-Asn was obtained from Sigma-Aldrich, (Milwaukee, WI, USA). NADH (disodium salt, grade II, $ 98%) and crystalline bovine serum albumin (fraction V) were purchased from Boehringer Mannheim (Mannheim, Germany). Nessler’s reagent and glutamate dehydrogenase were obtained from Fluka (Taufkirchen, Germany). All primers were synthe- sized and purified by MWG-biotech AG (Ebersberg, Germany). TOPO cloning kit and all other molecular biology reagents were from Invitrogen (Carlsbad, CA, USA). Directed evolution of L-ASNase Directed evolution of l-ASNase was carried out using the StEP [45]. Plasmids (pCR Ò T7 ⁄ CT-TOPO Ò ) containing the nucleotide sequences of l-ASNase from E. carotovora (Ecal-ASNase; NCBI accession number: AY560097, the enzyme was cloned as a non-tagged protein) [15] and E. chrysanthemi 3937 (ErL-ASNase; NCBI accession num- ber AY560098, the enzyme was cloned as a non-tagged pro- tein) [16] were used as parental sequences in the PCR. The forward primers used in the reaction were the 5¢-ATGGAACGATGGTTTAAATCTCTG-3¢ and 5¢-ATG TTTAACGCATTATTCGTTGTTGTTTTTG-3¢, and the reverse primers were the 5¢ -TCAATAGGTGTGGAAATA GTCCTGG-3¢ and 5¢-TTAAGCTTTTAATAAGCGTGG AAGTAATCC-3¢. The PCR was carried out in a total vol- ume of 50 lL containing 25 ng of each primer, 10 ng of template plasmid DNA, 0.2 mm of each dNTP, 5 lLof 10· Taq buffer, 1.5 mm MgCl 2 buffer and 2.5 units of Taq DNA polymerase (Stratagene, La Jolla, CA, USA). The PCR procedure comprised 99 cycles of 30 s at 94 °C and 10 s at 46 °C. Fig. 8. Structural representations of the mutant enzymes. A closer view of the mutation site of Asp133Thr, (A); Asp133Ile, (B); and Asp133Leu, (C). Hydrogen bonds are shown as dashed lines and residues are labelled. The models of the mutated enzymes were constructed using WHAT IF [51]. The figure was created using PYMOL [54]. G. A. Kotzia and N. E. Labrou Engineering thermal stability of L-asparaginase FEBS Journal 276 (2009) 1750–1761 ª 2009 The Authors Journal compilation ª 2009 FEBS 1757 Cloning, expression and screening for thermostable mutants Following completion of StEP, the resulting PCR amplicon was treated with the restriction enzyme DpnI to eliminate parental plasmid DNA and was TOPO ligated to pCR Ò T7 ⁄ CT-TOPO Ò expression vector. The presence of the stop codon in the 5¢-end of the reverse primers allowed the expression of non-tagged enzyme variants. The resulting expression constructs pT7Mut-ASNase were used to trans- form competent BL21(DE3)pLysS E. coli cells. E. coli cells, harbouring plasmids pT7MutASNase, were grown at 37 °C in 30 mL of Luria–Bertani medium containing 100 lgÆmL )1 ampicillin and 34 lg Æ mL )1 chloramphenicol. The synthesis of l-ASNases was induced by the addition of 1 m m isopro- pyl thio-b-d-galactoside when the absorbance at 600 nm was 0.6–0.8. Five hours after induction, cells were harvested by centrifugation at 4000 g and 4 °C for 20 min. In order to find thermotolerant mutants, cell-free extracts were incu- bated at 55 °C for 7.5 min and were then used to measure the residual activities using the coupled enzyme assay method described below. Saturation mutagenesis, library creation and screening Saturation mutagenesis at amino acid position 133 was per- formed by overlap extension using PCR [46]. Mutations were introduced using a set of degenerate synthetic oligonu- cleotides, in which the mutation site was diversified using a randomized NNN codon. The pairs of oligonucleotide primers used in the PCR for the saturation mutagenesis were as follows: the first pair 5¢-ATGGAACGATG GTTTAAATCTCTG-3¢ (P 1 ) and 5¢-GTGAAAAGCNNN AAGCCGGTAGTG-3¢ (P 2 ), and the second pair 5¢-CACT ACCGGCTTNNNGCTTTTCAC-3¢ (P 3 ) and 5¢-TCAAT AGGTGTGGAAATAGTCCTGG-3¢ (P 4 ). Sites of muta- tion are indicated in italics. The expression construct encod- ing the wild-type ErL-ASNase [16] was used as template DNA. After completion of the PCR (using primers P1, P2 and P3, P4), the PCR products were digested with DpnIto eliminate parental DNA, and were then used in another PCR as templates using P 1 and P 4 primers to amplify the entire mutated gene. The latter was TOPO ligated into a T7 expression vector (pEXP5-CT ⁄ TOPO Ò ) and recombinant plasmids were isolated and were used to transform compe- tent BL21(DE3)pLysS E. coli cells. E. coli cells were grown at 37 °C in 30 mL of Luria–Bertani medium containing 100 lgÆmL )1 ampicillin and 34 lgÆmL )1 chloramphenicol. The synthesis of the mutated enzymes was induced by the addition of 1 mm isopropyl thio-b-d-galactoside when the absorbance at 600 nm was 0.6–0.8. Four hours after induc- tion, cells were harvested by centrifugation at 4000 g and 4 °C for 20 min. Hundreds of transformants of the library were examined for their thermotolerance at 60 °C for 5 min, in order to identify promising mutants showing increased thermotolerance compared with the Asp133Val. The thermotolerance was estimated by measuring the residual activities using the coupled enzyme assay method described below. For the variants showing ‡ 45% residual activity, the mutations were determined by DNA sequencing. Purification of the wild-type and mutant enzymes Purification of the wild-type enzymes was carried out according to published methods [15,16]. The purification of mutants was accomplished by a two-step procedure, comprising a negative purification step using a DEAE– Sepharose CL6B, followed by an immobilized metal che- late-affinity chromatography on a Ni-NTA column. This was carried out as follows: cell paste was suspended in potassium phosphate buffer (5 mm, pH 7.5), sonicated and centrifuged at 10 000 g for 5 min. The supernatant was col- lected and applied to DEAE–Sepharose CL6B (1 mL, 0.5 · 2 cm i.d.) column, previously equilibrated with potas- sium phosphate buffer (5 mm, pH 7.5). Nonadsorbed pro- tein was washed off with 3 mL equilibration buffer. The flow-through and the first fraction (1 mL) of the washing step were mixed, adjusted to pH 8 (with the addition of 0.1 m potassium phosphate buffer, pH 8) and 0.1 m NaCl (by the addition of 5 m NaCl) before being applied to Ni-NTA (0.5 mL, 0.5 · 1 cm i.d.) column. The column was previously equilibrated with potassium phosphate buffer (50 mm, pH 8) containing 0.3 m NaCl. Nonadsorbed protein was washed off with 8 mL equilibration buffer, followed by 8 mL potassium phosphate buffer (50 mm, pH 7.5) containing 0.3 m NaCl, and 8 mL potassium phos- phate buffer (50 mm, pH 7) containing 0.3 m NaCl. Bound l-ASNase was eluted with potassium phosphate buffer (50 mm, pH 6.2) containing 0.3 m NaCl. Collected fractions (2 mL) were assayed for l-asparaginase activity and pro- tein. Following purification, the wild-type enzyme as well as the mutant enzymes were dialysed against 1000 vol. of 0.1 m Tris ⁄ HCl pH 8.0 (for kinetic analysis; see below) or against 10 mm KH 2 PO 4 buffer pH 7 (for thermal inactiva- tion studies; see below). Assay of enzyme activity and protein Enzyme assays were performed at 37 °C at a Hitachi U-2000 double beam UV ⁄ Vis spectrophotometer carrying a thermostated cell holder (10 mm path length). Activities were measured by determining the rate of ammonia forma- tion, by coupling with glutamate dehydrogenase, according to Balcao et al. [47]. The final assay volume of 1 mL con- tained 71 mm Tris ⁄ HCl buffer, pH 8.0, 1 mm Asn, 0.15 mm a-ketoglutaric acid, 0.15 mm NADH, 4 units glutamate dehydrogenase and sample containing l-ASNase activity. Alternatively, the rate of ammonia formation was measured Engineering thermal stability of L-asparaginase G. A. Kotzia and N. E. Labrou 1758 FEBS Journal 276 (2009) 1750–1761 ª 2009 The Authors Journal compilation ª 2009 FEBS at 37 °C using the Nessler’s reagent [48]. One unit of l-ASNase activity is defined as the amount of enzyme that liberates 1 lmol of ammonia from l-Asn per min at 37 °C. Protein concentrations were determined at 25 °C using the method of Bradford [49] using BSA (fraction V) as standard. Kinetic analysis Steady-state kinetic measurements were performed as described previously [15,16,50]. The kinetic parameters k cat and K m were calculated by nonlinear regression analysis of experimental steady-state data. Turnover numbers were cal- culated on the basis of one active site per subunit. Kinetic data were analysed using the computer program grafit (Erythacus Software Ltd, Staines, UK) [34]. Molecular modelling and computational analysis Molecular modelling for creating the model of E. chrysant- hemi l-ASNase was carried out as described by Kotzia & Labrou [16]. The molecular modelling program what if [51] was used to predict the conformation of the mutant enzymes. Prediction of the conformation of the new side chains was performed as described by Chinea et al. [52] Poisson–Boltzmann electrostatic potential analysis of the mutant and the wild-type enzymes was carried out using swiss-pdb viewer [53]. The parameters utilized to calculate the electrostatic potential were: dielectric constant (protein) 4, dielectric constant (solvent) 80, solvent ionic strength 0.00, partial charged ‘on’. Nonbonded interactions were analysed by moltalk (http://i.mol.talk.org). The program pymol was used for inspection of models and crystal struc- tures [54]. Thermal stability of the wild-type, Asp133Val and saturation variants Thermal inactivation of the wild-type and Asp133Val and saturation variants was monitored by activity measure- ments. Samples of the enzymes, in 10 mm KH 2 PO 4 buffer pH 7, were incubated at a range of temperatures from 30 to 70 °C for 7.5 min. Subsequently, the samples were assayed for residual activity, using the coupled enzyme assay method described above. The T m values were deter- mined from the plots of relative inactivation (%) versus temperature (°C). The T m value is the temperature at which 50% of the initial enzyme activity is lost after heat treatment. The kinetics of thermal inactivation of the wild-type and Asp133Val was monitored at 50 °C. The kinetics of thermal inactivation of saturation variants was monitored at 57.5 °C. The rates of inactivation were followed by periodically removing samples for assay of enzymatic activity. Observed rates of inactivation (k in ) were deduced from plots of log (% remaining activity) versus time. Guanidinium chloride induced subunit dissociation Guanidinium chloride induced dissociation of the wild-type and mutant Asp133Val enzymes was carried out according to Shifrin et al. [32] in 50 mm KH 2 PO 4 pH 7.5. Guanidinium chloride treatments were performed in the presence of 3 m guanidinium chloride and measurements were taken for 5 min. The rate of dissociation was determined by the increase of the absorbance at 288 nm as described by Shifrin et al. [32]. Electrophoresis SDS ⁄ PAGE was performed according to the method of Laemmli [55] on a slab gel containing 12.5% (w ⁄ v) poly- acrylamide (running gel) and 2.5% (w ⁄ v) stacking gel. The protein bands were stained with Coomassie Brilliant Blue R-250. Acknowledgements This work was financially supported by the Hellenic General Secretariat for Research and Technology: Operational Program for Competitiveness, Joint Research and Technology Program. 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Labrou Laboratory of Enzyme Technology, Department of Agricultural. function of time in 0.05 M phosphate buffer, pH 7.5, containing 3 M guanidine chloride. Changes in absorbance at 288 nm were monitored for 5 min. Engineering thermal stability of L-asparaginase. Enhancing the stability of l-ASNase by protein engineering improves its body-residence time, and thereby minimizes immunosuppressive effects by lower- ing the therapeutic dose. After one round of directed evolution