Characterization of a Type II L-Asparaginase from the Halotolerant Bacillus subtilis CH11

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Characterization of a Type II L-Asparaginase from the Halotolerant Bacillus subtilis CH11 © 2023 by the authors. Licensee MDPI, Basel, Switzerland Published version Arredondo-Nuñez, Annsy; Monteiro, Gisele; Flores-Fernández, Carol N.; Antenucci, Lina; Permi, Perttu; Zavaleta Amparo, Iris Arredondo-Nuñez, A., Monteiro, G., Flores-Fernández, C. N., Antenucci, L., Permi, P., & Zavaleta Amparo, I. (2023). Characterization of a Type II L-Asparaginase from the Halotolerant Bacillus subtilis CH11. Life, 13(11), Article 2145. https://doi.org/10.3390/life13112145 2023 Citation: Arredondo-Nuñez, A.; Monteiro, G.; Flores-Fernández, C.N.; Antenucci, L.; Permi, P.; Zavaleta, A.I. Characterization of a Type II L-Asparaginase from the Halotolerant Bacillus subtilis CH11. Life 2023,13, 2145. https://doi.org/ 10.3390/life13112145 Academic Editors: Martina Aulitto and Giovanni Gallo Received: 29 September 2023 Revised: 28 October 2023 Accepted: 30 October 2023 Published: 31 October 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). life Article Characterization of a Type II L-Asparaginase from the Halotolerant Bacillus subtilis CH11 Annsy Arredondo-Nuñez 1, Gisele Monteiro 2, Carol N. Flores-Fernández 1,*, Lina Antenucci 3, Perttu Permi 3,4,5 and Amparo Iris Zavaleta 1,* 1 Laboratorio de Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad Nacional Mayor de San Marcos, Lima 01, Peru; annsy[email protected] 2 Department of Pharmaceutical and Biochemical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo 05508-000, Brazil; [email protected] 3Department of Biological and Environmental Science, Nanoscience Center, University of Jyvaskyla, P.O. Box 35, FI-40014 Jyvaskyla, Finland; [email protected] (L.A.); [email protected] (P.P.) 4Department of Chemistry, Nanoscience Center, University of Jyvaskyla, P.O. Box 35, FI-40014 Jyvaskyla, Finland 5Institute of Biotechnology, Helsinki Institute of Life Science, University of Helsinki, P.O. Box 65, FI-00014 Helsinki, Finland *Correspondence: [email protected] (C.N.F.-F.); [email protected] (A.I.Z.) Abstract: L-asparaginases from bacterial sources have been used in antineoplastic treatments and the food industry. A type II L-asparaginase encoded by the N-truncated gene ansZP21 of halotolerant Bacillus subtilis CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in Escherichia coli BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited an activity of 234.38 U mg −1 and a maximum catalytic activity at pH 9.0 and 60 ◦ C. The enzyme showed a homotetrameric form with an estimated molecular weight of 155 kDa through gel filtration chromatography. The enzyme half-life at 60 ◦ C was 3 h 48 min, and L-asparaginase retained 50% of its initial activity for 24 h at 37 ◦ C. The activity was considerably enhanced by KCl, CaCl 2 , MgCl 2 , mercaptoethanol, and DL-dithiothreitol (p-value < 0.01). Moreover, the V max and K m were 145.2 µ mol mL −1 min −1 and 4.75 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications. Keywords: L-asparaginase II; Bacillus subtilis; saline environment; biochemical characterization 1. Introduction L-asparaginase (EC 3.5.1.1) hydrolyzes L-asparagine into aspartic acid and ammonia via an intermediate beta-acyl-enzyme [ 1 , 2 ]. This well-known enzyme is used in cancer therapy, such as childhood acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphoma, and lymphoid system disorders [ 3 ]. The antineoplastic action of L-asparaginase occurs because cancer cells are not able to synthesize enough L-asparagine; depletion of this compound in serum causes the death of cancer cells. However, several side effects have been reported in patients treated with the available L-asparaginases in the market [ 4 , 5 ], such as enzymes from Escherichia coli and Erwinia chrysanthemi, which can present up to 5% of L-glutaminase activity and clinical resistance of tumor cells during antineoplastic treatment caused by the immune response to the drug from bacterial origin [ 6 , 7 ]. In addition, L- asparaginase has frequently been used in the food industry to reduce the formation of carcinogenic acrylamide, which is generated during heat treatment, and improve taste and nutritional contributions in food [8]. The sources for obtaining this protein are diverse, including plants, animals, bacteria, fungi, and yeasts. Nevertheless, bacterial sources are the most interesting and more comprehensively studied because of their easy handling and genetic manipulation, rapid growth, lower genome complexity, and economically viable production cost [ 9 , 10 ]. Bacteria Life 2023,13, 2145. https://doi.org/10.3390/life13112145 https://www.mdpi.com/journal/life Life 2023,13, 2145 2 of 12 L-asparaginases are divided into two main classes: type I, which are cytosolic enzymes with low affinity to L-asparagine (K m in mM extent); and type II, which are periplasmic enzymes with high affinity to L-asparagine (K m in µ M limits). The K m kinetic parameter is important to direct the industrial enzyme application. Enzymes with high affinity to the substrate are required for human cancer therapy because bloodstream L-asparagine concentrations are in µ M range; however, in the food industry, thermostability is the main feature to be considered [11]. On the other hand, L-asparaginases from bacteria isolated from hypersaline environments, especially from the Bacillus genus, have been described as the most promising anticancer compounds as they show lower immune responses and higher activity [ 12 – 14 ]. The genome of Bacillus subtilis (B. subtilis) has two genes encoding for L-asparaginase (ansA and ansZ). The ansA gene encodes L-asparaginase I, an intracellular protein with low affinity to the substrate. In contrast, the ansZ gene encodes L-asparaginase II, an extracellular enzyme with higher substrate affinity relevant for clinical safety in treatments with L-asparaginase [15,16]. Native L-asparaginases are not highly expressed even by optimizing the growth medium; therefore, the usage of a heterologous expression system represents a great tool for high protein expression, simplicity of purification using specific tags, and ultimately, biochemical, structural, and biophysical studies. Moreover, current studies aim to optimize production media [ 10 , 17 ] and to obtain higher yields of purified protein via different strategies, e.g., including a signal peptide, optimizing a promoter to obtain extracellular proteins [ 18 , 19 ], and truncating the N-terminus of L-asparaginase [ 15 , 20 ]; overall, the target is to obtain better quality, efficiency, and safety of the L-asparaginase. Studies on type II recombinant proteins from Bacillus sp. have been performed to obtain new sources for this pharmaceutically and biotechnologically important protein to meet industrial demands. Thus, several groups have reported the outstanding characteristics of L-asparaginases in terms of thermostability [ 8 ], high substrate affinity [ 2 , 21 ], enzymatic activity, and purity [ 22 , 23 ]. However, native and recombinant L-asparaginases might have limitations in their therapeutic effect and intrinsic half-life; therefore, further studies are required in the pharmaceutical setting in combination with nanoparticle systems [24]. This paper describes the cloning, heterologous expression, and purification of N- terminally truncated type II L-asparaginase of B. subtilis CH11 from Chilca salterns in Lima, Peru. We have characterized its thermostability in addition to the contribution of temperature, pH, and co-factors to the enzymatic activity. We also determined the kinetic parameters of the enzyme. 2. Materials and Methods 2.1. Bacteria Strains, Medium, and Chemicals Bacillus subtilis CH11 strain isolated from Chilca Salterns in Lima, Peru, belonged to the collection of the Molecular Biology Laboratory, Faculty of Pharmacy and Biochemistry, Universidad Nacional Mayor de San Marcos. This strain was conserved at − 80 ◦ C in TSB medium/glycerol 30% (v/v). TSB medium (g/L): casein peptone, 17; K 2 HPO 4 , 2.5; glucose, 2.5; NaCl, 5; soya peptone, 3; pH 7.3. LB-Miller (g/L): yeast extract, 5; peptone from casein, 10; NaCl, 10; pH 7.0. T4 DNA ligase, Phusion DNA polymerase, and Escherichia coli BL21(DE3)pLysS were purchased from Thermo Scientific ® (Waltham, MA, USA). Restriction endonucleases were obtained from New England Biolabs ® (Ipswich, MA, USA). The QIAprep ® Spin Miniprep Kit was acquired from QIAGEN (Hilden, Germany). pET-15b and BugBuster ® Master Mix were from Novagen ® (Merck—Darmstadt, Germany). Finally, the Bicinchoninic Acid Kit and Isopropyl β -D-thiogalactopyranoside were procured from Sigma-Aldrich®(St. Louis, MO, USA). 2.2. Bioinformatic Analysis The native ansZ gene sequence was analyzed through the SignalP—6.0 server (DTU Health Tech, Kgs. Lyngby, Denmark) to identify the presence of signal peptide. The Life 2023,13, 2145 3 of 12 molecular weight and isoelectric point were predicted using ProtParam (SIB Bioinformatics Resource, Lausanne, Switzerland). The monomer’s structure was predicted using AlphaFold2 (EMBL-EBI, Cambridge, UK). 2.3. Cloning of the ansZP21 Gene Encoding L-ASNasaZP21 The B. subtilis CH11 strain was grown in TSB medium for 24 h at 37 ◦ C. The genomic DNA was extracted according to Montes et al. [ 25 ]. The ansZ gene without the signal peptide YccC (first 60 base pairs), denominated as ansZP21, was amplified by PCR from the extracted DNA of Bacillus sp. CH11 using the forward primer 5 0 -TTT CAT ATG CCA CAT TCT CC T GAA ACA AAA GAA TCC CC-3 0 and the reverse primer 5 0 -TGC C GG ATC C TC AAT ACT CAT TGA AAT AAG C-3 0 . The gene was cloned using the restriction enzymes NdeI and BamHI, whose recognition sequences are in bold in the primers detailed above. PCR was carried out using Phusion DNA polymerase (2 U µ L −1 ); the reaction conditions were an initial denaturation at 98 ◦ C for 30 s, followed by 35 cycles at 98 ◦ C for 10 s, 58 ◦ C for 30 s, 72 ◦ C for 20 s, and a final extension at 72 ◦ C for 5 min (T100 Thermal Cycler, Bio-Rad, Hercules, CA, USA). The PCR products were cloned into pET-15b using 1 U of T4 DNA ligase and transformed into Escherichia coli DH5 α . Then, the plasmids were extracted using kit QIAprep ® Spin Miniprep Kit and sent for sequencing to confirm the correct cloning of the ansZP21 gene. The correct expression vector was transformed into Escherichia coli BL21(DE3)pLysS host cells. 2.4. Expression and Purification of L-ASNasaZP21 Escherichia coli BL21(DE3)pLysS cells were used for protein expression. Cells were grown on 500 mL LB-Miller medium supplemented with 100 µ g mL −1 ampicillin at 37 ◦ C, 230 rpm on an orbital shaker. The protein expression was induced by adding Isopropyl β -D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM when the OD 600 reached 0.6. Post-induction, the culture was incubated for 14 h at 22 ◦ C and 230 rpm. Subsequently, the cells were harvested by centrifugation at 2133 g for 20 min at 4 ◦ C; the pellet was washed with 1X PBS buffer (pH 7.4) and disrupted using BugBuster ® Master Mix reagent, following the manufacturer’s instructions. The clarified lysate containing 6X-His-tagged L-ASNasaZP21 was recovered by centrifugation at 12,555 g for 30 min at 4 ◦ C and used for purification by immobilized metal affinity chromatography (IMAC) using an FPLC system (ÄKTA start, GE Healthcare, Chicago, IL, USA). Briefly, the clarified lysate in 50 mM Tris-HCl containing 100 mM NaCl and 20 mM imidazole, pH 8.5, was loaded onto a pre-equilibrated HisTrap TM FF column of 5 mL (GE Healthcare) at a flow rate of 1 mL min −1 . Unbound proteins were eliminated by washing the column with a 5 column volume (CV) of the buffer. Finally, the enzyme was eluted by a linear gradient of imidazole (up to 500 mM), desalted in Tris-HCl pH 8.5, and stored at 4 ◦C for further analysis. 2.5. Molecular Weight Determination The molecular weight of the purified L-ASNasaZP21 was determined by size exclusion chromatography using a HiPrep TM 16/60 Sephacryl ® S-200 HR column (GE Healthcare) and 50 mM Tris-HCl containing 100 mM NaCl, pH 8.5, at a flow rate of 0.5 mL min −1 . The standard curve was derived using a Protein Standard Mix, 15–600 kDa (Sigma-Aldrich ® , St. Louis, MO, USA), composed of ρ -aminobenzoic acid (0.14 kDa), ribonuclease A type I-A (13.7 kDa), grade VI albumin (44.3 kDa), γ -globulin (150 kDa), and thyroglobulin (670 kDa). The molecular weight was estimated on a semi-log graph following the method described by Mahajan et al. [26]. 2.6. SDS-PAGE and Zymography The purity fraction of the L-ASNasaZP21 was evaluated by SDS-PAGE using β - mercaptoethanol as a reducing agent. Zymography was utilized to assess the L-asparaginase activity in situ following electrophoresis with 5% polyacrylamide gel. The gel was incubated in a solution containing 25 mL of 50 mM Tris-HCl pH 8.6, 2 mL of 189 mM L-asparagine, 2 mL of 2 M hydroxylamine, and 1.6 mL of 2 M NaOH. The incubation was performed at Life 2023,13, 2145 4 of 12 37 ◦ C for 20 min in a Mini Rocker Platform (Bio-Rad). Finally, the gel was stained with a solution containing 10% FeCl 2 , 5% trichloroacetic acid (TCA), and 0.66 M HCl, which enabled visualization of a positive reaction based on the L-aspartic acid β -hydroxamate (AHA) colorimetric assay [27]. 2.7. L-asparaginase Activity and Protein Assay The L-asparaginase activity was evaluated using Nessler’s method, with some modifications [ 28 ]. The reaction consisted of 100 µ L of 50 mM Tris-HCl pH 8.6, 10 µ L of 189 mM L-asparagine, 90 µ L of H 2 O, and 10 µ L of sample. This mixture was incubated for 10 min at 37 ◦ C and stopped using 10 µ L of 1.5 M trichloroacetic acid. A volume of 25 µ L of the previous reaction was mixed with 25 µ L of Nessler solution and 200 µ L of H 2 O, and the released free ammonia was quantified, measuring the absorbance at 436 nm. In the negative control, H 2 O was used instead of the enzyme, and the reaction was stopped for the blank before adding the enzyme. A standard calibration curve was derived using different known concentrations of ammonium sulfate between 0.005 and 0.109 µ moles. One unit of enzyme (U) produces 1.0 µ mole of ammonia from L-asparagine per minute under optimum conditions. Protein concentrations were measured according to the Bicinchoninic Acid Kit for 96-well plate-assays, following the manufacturer’s instructions. Bovine serum albumin (BSA) (Sigma-Aldrich ® , St. Louis, MO, USA) was used as a standard at intervals within 80 to 800 µg of protein. 2.8. Biochemical Characterization The temperature effect on L-ASNasaZP21 activity was investigated between 22 and 80 ◦ C at a fixed pH equal to 8.6. The pH effect was evaluated between 3.0 and 10.0 using appropriate buffers: pH 3.0–5.0, 50 mM sodium citrate, pH 6.0–7.0, 50 mM sodium phosphate, pH 8.0–9.0, 50 mM Tris-HCl buffer, pH 10.0, sodium bicarbonate-NaOH. The temperature was fixed at 60 ◦C. The results were expressed as the relative activity (%). The half-life of L-ASNasaZP21 at 22, 37, and 60 ◦ C was determined by incubation from 1 to 24 h, and the residual activity was measured at 60 ◦ C for 10 min via the Nessler method with modifications described above; a control sample without incubation was used. The reaction rate describing heat inactivation was calculated by plotting the time (h) along the x-axis vs. the logarithmic residual activity along the y-axis. The inactivation rate constant (k) was estimated using linear regression [29]: ln [A]t=−kt + ln [A]0(1) where [A] 0 is the control activity (100%) and the [A] t is the activity at an indicated time, t (h). The half-life was determined through the following equation [30]: t(1/2) = ln(2)k−1(2) The effect of inhibitors and ions was examined following the same protocol described above and supplementing the standard reaction mixture with appropriate inhibitors and salts. The tested inhibitors were PMSF, Urea, Mercaptoethanol, DL-Dithiothreitol, SDS, and EDTA at final concentrations of 10 mM, and Glutathione at final concentration of 5 mM. The tested salts with mono and divalent cations were NaCl, KCl, CaCl 2 , MgCl 2 , MnCl 2 , BaCl 2 , CuCl 2 , and CoCl 2 , all at final concentrations of 100 mM. The enzyme activity was expressed as the relative activity (%) compared with the control without any supplemented component. The kinetic assay reactions were carried out at pH 9, 60 ◦ C, and with an incubation time of 10 min. The substrate was tested in a concentration range from 2 to 14 mM. The Vmax and Kmvalues were calculated via the Lineweaver–Burk plot. 2.9. Data Collection and Analysis All the analyses were carried out in duplicate and expressed as the mean ± the standard deviation (SD). Data were evaluated using one-way ANOVA and Dunnett’s Life 2023,13, 2145 5 of 12 multiple comparison test using GraphPad Prism version 10.0.2. software (San Diego, CA, USA), with significance defined as p< 0.01. 3. Results and Discussion 3.1. Cloning of the ansZP21 Gene and Sequence Analysis The lipoprotein signal peptide type II YccC reported in B. subtilis was found to be in the N-terminal amino sequence of the ansZ gene in the present study [8,31]. This contains a conserved Cys20 residue that forms the cleavage site; studies have suggested that the formation of the mature protein is involved in proper folding, where post-translational diacylglycerol modification of the Cys residue is required for signal peptide release [15,32,33] . In addition, Onishi et al. [ 15 ] reported that E. coli might not process the signal peptide of L-asparaginase from Bacillus sp., resulting in incorrect protein folding and a lower purification yield and purity. The native ansZ encoded protein included a signal peptide identified via bioinformatics analysis on the SignalP—6.0 server with a probability of 0.996% (Figure 1a). This finding is in line with the AlphaFold2 structure prediction of L-asparaginase II from B. subtilis (Figure 1b) [ 34 , 35 ]. Therefore, signal peptides between 1 and 19 amino acid residues and Cys20 residue were removed when cloning the protein for E. coli expression. Based on that, the ansZP21 gene has 1068 bps, encoding the protein L-ASNasaZP21 of 355 amino acids, with a molecular weight and isoelectric point of 37.91 kDa and 6.16, respectively. Life 2023, 13, x FOR PEER REVIEW 5 of 13 was expressed as the relative activity (%) compared with the control without any supplemented component. The kinetic assay reactions were carried out at pH 9, 60 °C, and with an incubation time of 10 min. The substrate was tested in a concentration range from 2 to 14 mM. The Vmax and Km values were calculated via the Lineweaver–Burk plot. 2.9. Data Collection and Analysis All the analyses were carried out in duplicate and expressed as the mean ± the standard deviation (SD). Data were evaluated using one-way ANOVA and Dunnett’s multiple comparison test using GraphPad Prism version 10.0.2. software (San Diego, CA, USA), with significance defined as p < 0.01. 3. Results and Discussion 3.1. Cloning of the ansZP21 Gene and Sequence Analysis The lipoprotein signal peptide type II YccC reported in B. subtilis was found to be in the N-terminal amino sequence of the ansZ gene in the present study [8,31]. This contains a conserved Cys20 residue that forms the cleavage site; studies have suggested that the formation of the mature protein is involved in proper folding, where post-translational diacylglycerol modification of the Cys residue is required for signal peptide release [15,32,33]. In addition, Onishi et al. [15] reported that E. coli might not process the signal peptide of L-asparaginase from Bacillus sp., resulting in incorrect protein folding and a lower purification yield and purity. The native ansZ encoded protein included a signal peptide identified via bioinformatics analysis on the SignalP—6.0 server with a probability of 0.996% (Figure 1a). This finding is in line with the AlphaFold2 structure prediction of L-asparaginase II from B. subtilis (Figure 1b) [34,35]. Therefore, signal peptides between 1 and 19 amino acid residues and Cys20 residue were removed when cloning the protein for E. coli expression. Based on that, the ansZP21 gene has 1068 bps, encoding the protein L-ASNasaZP21 of 355 amino acids, with a molecular weight and isoelectric point of 37.91 kDa and 6.16, respectively. (a) Life 2023, 13, x FOR PEER REVIEW 6 of 13 (b) Figure 1. Signal peptide prediction by SignalP—6.0 of ansZ gene. (a) AlphaFold structure prediction of native ansZ gene. (b) Signal peptide, disordered N-terminal, and structural parts are shown in red, blue, and green, respectively. 3.2. Expression and Purification of L-ASNasaZP21 B. subtilis L-ASNasaZP21 expressed in heterologous E. coli BL21(DE3)pLysS and purified showed a specific activity of 234.38 U mg −1 , which is higher than reported values in similar proteins [15,36,37]. This may result from the better protein solubility and reduced misfolding associated with N-terminal truncation and the optimized expression protocol [20,38]. In addition, Moura et al. [20] reported that E. coli BL21(DE3)pLysS (89.0 ± 4.4) expresses L-asparaginase with a higher enzymatic activity compared with other E. coli strains (T7 Express Crystal, 57.0 ± 1.7; Tuner (DE3), 41.6 ± 2.0; C43 (DE3), 22.4 ± 1.6; BL21 (DE3), 12.5 ± 1.2; Lemo21 (DE3), 10.9 ± 1.2; SHuffle T7, 4.9 ± 1.9; GroEL (DE3), 2.2 ± 2.1). Thus, it is presumed that E. coli strains Tuner (DE3), C43 (DE3), Lem21 (DE3), and (DE3) are not efficient hosts for adequate protein folding. Furthermore, SHuffle T7 does not favor the disulfide bond between Cys99 and Cys127 bonds for the correct structural conformation, and GroEL presents chaperones with no activity at low post-induction temperatures [20]. A purification factor of 85.2-fold and a recovery yield of 61.9% were achieved after the affinity chromatography (Table 1). The N-truncated version of our L-asparaginase was expressed, including an N-terminal 6X-His-tag, which allowed high selectivity to obtain a highly purified protein from a complex sample [8,39]. Studies on other type II L-aspar- aginases from Bacillus sp. using affinity chromatography have been reported, achieving activities of 4438.6 U mg −1 [22], 1146 U mg −1 [40], and 162.9 U mg −1 with a recovery yield of 67.21% [18] (Table 2). Specific activity (U mg −1 ) is clinically relevant because the patient will receive an L-asparaginase dose based on units per body surface area. In clinical terms, this means that a much higher amount of protein (in mg) is necessary to reach therapeutic efficacy for enzymes with low specific activity. In this context, L-ASNasaZP21 has specific activity very similar to that applied in clinical practice [11]. Table 1. Summary of the purification of L-ASNasaZP21. Total Activity (U) Total Protein (mg) Activity (U mg −1 ) Purification-Fold Yield (%) Crude extract 2.38 × 10 2 86.39 2.75 1.0 100.0 Ni-affinity 1.47 × 10 2 0.63 234.38 85.2 61.9 Figure 1. Signal peptide prediction by SignalP—6.0 of ansZ gene. ( a ) AlphaFold structure prediction of native ansZ gene. ( b ) Signal peptide, disordered N-terminal, and structural parts are shown in red, blue, and green, respectively. Life 2023,13, 2145 6 of 12 3.2. Expression and Purification of L-ASNasaZP21 B. subtilis L-ASNasaZP21 expressed in heterologous E. coli BL21(DE3)pLysS and purified showed a specific activity of 234.38 U mg −1 , which is higher than reported values in similar proteins [ 15 , 36 , 37 ]. This may result from the better protein solubility and reduced misfolding associated with N-terminal truncation and the optimized expression protocol [ 20 , 38 ]. In addition, Moura et al. [ 20 ] reported that E. coli BL21(DE3)pLysS ( 89.0 ±4.4 ) expresses L-asparaginase with a higher enzymatic activity compared with other E. coli strains (T7 Express Crystal, 57.0 ± 1.7; Tuner (DE3), 41.6 ± 2.0; C43 (DE3), 22.4 ± 1.6; BL21 (DE3), 12.5 ± 1.2; Lemo21 (DE3), 10.9 ± 1.2; SHuffle T7, 4.9 ± 1.9; GroEL (DE3), 2.2 ± 2.1). Thus, it is presumed that E. coli strains Tuner (DE3), C43 (DE3), Lem21 (DE3), and (DE3) are not efficient hosts for adequate protein folding. Furthermore, SHuffle T7 does not favor the disulfide bond between Cys99 and Cys127 bonds for the correct structural conformation, and GroEL presents chaperones with no activity at low post-induction temperatures [20]. A purification factor of 85.2-fold and a recovery yield of 61.9% were achieved after the affinity chromatography (Table 1). The N-truncated version of our L-asparaginase was expressed, including an N-terminal 6X-His-tag, which allowed high selectivity to obtain a highly purified protein from a complex sample [ 8 , 39 ]. Studies on other type II L-asparaginases from Bacillus sp. using affinity chromatography have been reported, achieving activities of 4438.6 U mg −1 [ 22 ], 1146 U mg −1 [ 40 ], and 162.9 U mg −1 with a recovery yield of 67.21% [ 18 ] (Table 2). Specific activity (U mg −1 ) is clinically relevant because the patient will receive an L-asparaginase dose based on units per body surface area. In clinical terms, this means that a much higher amount of protein (in mg) is necessary to reach therapeutic efficacy for enzymes with low specific activity. In this context, L- ASNasaZP21 has specific activity very similar to that applied in clinical practice [11]. Table 1. Summary of the purification of L-ASNasaZP21. Total Activity (U) Total Protein (mg) Activity (U mg−1) Purification-Fold Yield (%) Crude extract 2.38 ×10286.39 2.75 1.0 100.0 Ni-affinity 1.47 ×1020.63 234.38 85.2 61.9 Table 2. Physicochemical and kinetic characteristics of L-asparaginases of genus Bacillus. Source pH Temperature (◦C) Activity (U mg−1)Km(mM) Chromatography 1References Bacillus subtilis CH11 9.0 60 234.38 4.75 AC This study B. subtilis BDRD-ST26 - 65 162.90 5.29 HIC, IEX, GC Feng et al. [36] B. subtilis 168 8.0 7.5 65 50 45.40 31.90 2.06 7.06 IEX, HIC Onishi et al. [15] B. subtilis B11-06 7.5 40 92.45 0.43 HIC Jia et al. [37] B. subtilis KDPS1 5.0 37 - - IEX Sanghvi et al. [ 41 ] B. aryabhattai ITBHU02 8.5 40 680.50 0.25 HIC, GF Singh et al. [21] B. amyloliquefaciens MKSE 8.5 65 136.30 1.15 AC Yim et al. [8] B. cereus 9.0 50 550.80 9.38 HIC, IEX, GF Feng et al. [36] B. megaterium H-1 8.0 40 1146.29 21.63 AC Lu et al. [40] B. sonorensis 7.0 45 4438.62 2.00 AC Aly et al. [22] B. velenzensis 7.5 37 31.77 0.04 GF Mostafa et al. [2] -, not determined. 1AC (affinity), IEX (ion exchange), HIC (hydrophobic interaction), GC (gel filtration). 3.3. Molecular Weight Determination, SDS-PAGE, and Zymography SDS-page analysis showed that the molecular weight of L-ASNasaZP21 was 38 kDa, as expected from the bioinformatic analysis, and the purity grade was relatively high (Figure 2a). The zymography demonstrated the L-asparaginase activity in situ, although the molecular weight did not match that observed in denaturing conditions (Figure 2b). Life 2023,13, 2145 7 of 12 This is presumably associated with the oligomerization of the protein in native conditions. In accordance with this, the molecular weight determined by gel filtration chromatography was 155 kDa, indicating the possible tetrameric structure of L-ASNasaZP21, in agreement with preliminary studies [42,43]. Life 2023, 13, x FOR PEER REVIEW 8 of 13 (a) (b) Figure 2. L-ASNasaZP21 analysis by SDS-PAGE (a) and zymography (b). Lanes: M, PageRuler TM Prestained (Thermo Scientific ® , Waltham, MA, USA); 1, purified L-ASNasaZP21. (a) (b) Figure 3. Effect of temperature (a) and pH (b) on the enzymatic activity of L-ASNasaZP21. The relative activity was expressed as a percentage of the maximum activity. Error bars represent one standard deviation from the mean (n = 2). 3.5. Molecular Weight Determination, SDS-PAGE, and Zymography SDS-page analysis showed that the molecular weight of L-ASNasaZP21 was 38 kDa, as expected from the bioinformatic analysis, and the purity grade was relatively high (Figure 2a). The zymography exhibited the L-asparaginase activity in situ, although the molecular weight did not match that observed in denaturing conditions (Figure 2b). This is presumably associated with oligomerization of the protein in native conditions. In accordance with this, the molecular weight determined by gel filtration chromatography was 155 kDa, indicating the possible tetrameric structure of L-ASNasaZP21, in agreement with preliminary studies [42,43]. 3.6. Effect of Temperature and pH L-ASNasaZP21 exhibited an optimum activity at 60 °C (Figure 3a), 2.7-fold higher than at 37 °C. The enzyme retained more than 60% of its activity at 45 °C and around 30% at 70 °C. The optimum pH of L-ASNasaZP21 was 9.0 (Figure 3b), retaining more than 80% of its activity at physiological pH (pH 7). These results agree with those of Feng et al. [36], who reported an N-truncated L-asparaginase with an optimum temperature of 65 °C. Figure 2. L-ASNasaZP21 analysis by SDS-PAGE ( a ) and zymography ( b ). Lanes: M, PageRuler TM Prestained (Thermo Scientific®, Waltham, MA, USA); 1, purified L-ASNasaZP21. 3.4. Effect of Temperature and pH L-ASNasaZP21 exhibited optimum activity at 60 ◦ C (Figure 3a), 2.7-fold higher than at 37 ◦ C. The enzyme retained more than 60% of its activity at 45 ◦ C and around 30% at 70 ◦ C. The optimum pH of L-ASNasaZP21 was 9.0 (Figure 3b), retaining more than 80% of its activity at physiological pH (pH 7). These results agree with those of Feng et al. [36], who reported an N-truncated L-asparaginase with an optimum temperature of 65 ◦ C. However, this differed from that reported for other type II L-asparaginases from B. subtilis, which exhibited optimum activity at 40 ◦ C and pH 7.5 [ 37 ], as well as at 37 ◦ C and pH 5.0 [ 41 ]. These differences might be because the protein was from a halotolerant bacterium, in line with Lakshmi et al. [ 44 ]. Nevertheless, most bacterial L-asparaginases have shown optimum activity between 30 and 50 ◦C [45] and at pH between 7.0 and 9.0 [46]. Life 2023, 13, x FOR PEER REVIEW 8 of 13 (a) (b) Figure 2. L-ASNasaZP21 analysis by SDS-PAGE (a) and zymography (b). Lanes: M, PageRuler TM Prestained (Thermo Scientific ® , Waltham, MA, USA); 1, purified L-ASNasaZP21. (a) (b) Figure 3. Effect of temperature (a) and pH (b) on the enzymatic activity of L-ASNasaZP21. The relative activity was expressed as a percentage of the maximum activity. Error bars represent one standard deviation from the mean (n = 2). 3.5. Molecular Weight Determination, SDS-PAGE, and Zymography SDS-page analysis showed that the molecular weight of L-ASNasaZP21 was 38 kDa, as expected from the bioinformatic analysis, and the purity grade was relatively high (Figure 2a). The zymography exhibited the L-asparaginase activity in situ, although the molecular weight did not match that observed in denaturing conditions (Figure 2b). This is presumably associated with oligomerization of the protein in native conditions. In accordance with this, the molecular weight determined by gel filtration chromatography was 155 kDa, indicating the possible tetrameric structure of L-ASNasaZP21, in agreement with preliminary studies [42,43]. 3.6. Effect of Temperature and pH L-ASNasaZP21 exhibited an optimum activity at 60 °C (Figure 3a), 2.7-fold higher than at 37 °C. The enzyme retained more than 60% of its activity at 45 °C and around 30% at 70 °C. The optimum pH of L-ASNasaZP21 was 9.0 (Figure 3b), retaining more than 80% of its activity at physiological pH (pH 7). These results agree with those of Feng et al. [36], who reported an N-truncated L-asparaginase with an optimum temperature of 65 °C. Figure 3. Effect of temperature ( a ) and pH ( b ) on the enzymatic activity of L-ASNasaZP21. The relative activity was expressed as a percentage of the maximum activity. Error bars represent one standard deviation from the mean (n= 2). Life 2023,13, 2145 8 of 12 3.5. Molecular Weight Determination, SDS-PAGE, and Zymography SDS-page analysis showed that the molecular weight of L-ASNasaZP21 was 38 kDa, as expected from the bioinformatic analysis, and the purity grade was relatively high (Figure 2a). The zymography exhibited the L-asparaginase activity in situ, although the molecular weight did not match that observed in denaturing conditions (Figure 2b). This is presumably associated with oligomerization of the protein in native conditions. In accordance with this, the molecular weight determined by gel filtration chromatography was 155 kDa, indicating the possible tetrameric structure of L-ASNasaZP21, in agreement with preliminary studies [42,43]. 3.6. Effect of Temperature and pH L-ASNasaZP21 exhibited an optimum activity at 60 ◦ C (Figure 3a), 2.7-fold higher than at 37 ◦ C. The enzyme retained more than 60% of its activity at 45 ◦ C and around 30% at 70 ◦ C. The optimum pH of L-ASNasaZP21 was 9.0 (Figure 3b), retaining more than 80% of its activity at physiological pH (pH 7). These results agree with those of Feng et al. [ 36 ], who reported an N-truncated L-asparaginase with an optimum temperature of 65 ◦ C. However, these findings differed from those reported for other type II L-asparaginases from B. subtilis, which exhibited optimum activity at 40 ◦ C and pH 7.5 [ 37 ], as well as at 37 ◦ C and pH 5.0 [ 41 ]. These differences might be because the protein was from a halotolerant bacterium, in line with Lakshmi et al. [ 44 ]. Nevertheless, most bacterial L-asparaginases have shown optimum activity between 30 and 50 ◦C [45] and at pH between 7.0 and 9.0 [46]. 3.7. Effect of Metal Ions and Inhibitors The effects on enzymatic activity of inhibitors and ions are described in Table 3. The activity was slightly enhanced by KCl (1.2-fold) and MgCl 2 (1.5-fold), while the highest improvement in activity was observed in the presence of CaCl 2 (3.1-fold). This positive effect of ions on the activity has also been described for L-asparaginases from B. sonorensis [ 22 ] and B. amyloliquefaciens MKSE [ 8 ]. On the other hand, some authors have reported the inhibitory effect of MnCl2, CuCl2, and CoCl2on L-asparaginase activity [36,41]. Table 3. Effect of metal ions and inhibitors on L-ASNaseZP21 activity. Ions/Inhibitors Final Concentration Relative Activity (%) 1 Control - 100.00 KCl 100 mM 124.500 ±1.85 * NaCl 106.300 ±0.09 MgCl2149.800 ±4.04 * CaCl2310.700 ±3.28 * BaCl295.070 ±2.73 MnCl20.0 * CuCl20.0 * CoCl20.0 * PMFS 10 mM 118.700 ±5.77 Urea 96.260 ±7.36 Mercaptoethanol 139.800 ±3.52 * DL-dithiothreitol 271.100 ±37.00 * SDS 0.0 * EDTA 58.850 ±1.46 * Glutathione 5 mM 97.860 ±4.39 1 The relative activity was expressed as the percentage of activity compared with a control without metal ions. Error bars represent one standard deviation from the mean (n= 2). * p< 0.01 vs. control (ANOVA test). Likewise, L-ASNasaZP21 activity was enhanced in the presence of Mercaptoethanol (1.4-fold) and DL-dithiothreitol (2.7-fold). Reducing agents might decrease protein aggregation due to intermolecular disulfide bridge formation. These findings are similar to those with L-asparaginases from Pectobacterium carotovorum [ 47 ] and Erwinia carotovora [ 48 ]. The