Purification of Dickeya Solani L-Asparaginase and Study of the Influence of Tio2 and Zno Nanoparticles on Its Enzymatic Activity

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Puriﬁcation of Dickeya solanil-Asparaginase and Study of the Inﬂuence of TiO2 and ZnO Nanoparticles on Its Enzymatic Activity

Article in BioNanoScience · December 2019 DOI: 10.1007/s12668-019-00706-z

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Purification of Dickeya solani L-Asparaginase and Study of the Influence of TiO2 and ZnO Nanoparticles on Its Enzymatic Activity

Selma Allouache1,2 & Farida Bendali1 & Mohamed Azarkan3 & Toufik Mostefaoui2

# Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract L-asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartate and ammonia. In the present study, we describe the production, purification, and preliminary characterization of Dickeya solani L-asparaginase. The purification of L-asparaginase was done using ion-exchange chromatography on HiTrap Q-Sepharose Fast Flow followed by FPLC-gel filtration chromatography on Superdex 75 pg. The presence of the enzyme was confirmed by enzymatic activity measurements along with SDS- PAGE analysis. The purified L-asparaginase was shown to exhibit specificity towards L-asparagine, while being inactive against L-glutamine. This data allows us to suggest that the L-asparaginase from Dickeya solani might be clinically more relevant than, e.g., the L-asparaginase isolated from E. coli whichhydrolyzedalsoL-glutamine and produces L-glutamate, a neurotoxic agent.

We also demonstrated that the enzymatic activity was enhanced in the presence of TiO2 and ZnO nanoparticles, making them good candidates to improve L-asparaginase activity. Indeed, the results obtained show that the TiO2 nanoparticles increased the activity of L-asparaginase by a factor of 6.0 while the ZnO nanoparticles increased it twice.

Keywords Dickeya solani . Titanium(IV) oxide . Zinc oxide . L-asparaginase . Nanoparticles

1 Introduction proposed as a therapeutic agent against the pathogenic bacterium group A Streptococcus infections [4]. There is L-asparaginase (asparagine amide hydrolase, EC 3.5.1.1), a real need for L-asparaginase sources given its potential which catalyzes the hydrolysis of L-asparagine to L-aspar- industrial applications as food processing aid, as well as tate and ammonia, is a chemotherapeutic agent used for its interesting therapeutic applications [5]. against lymphoblastic leukemia [1, 2]. Being unable to Obtaining L-asparaginases from human and/or animal produce their own asparagine, leukemia cells were origin still remains challenging, making microorganisms destructed upon conversion of L-asparagine to aspartic better sources for the production and isolation of these acid and ammonia [3]. Recently, L-asparaginase was enzymes. The enzyme has been characterized from many bacterial genera [6]. In addition to E. coli [7], many other Electronic supplementary material The online version of this article microorganisms are capable of producing L-asparaginase (https://doi.org/10.1007/s12668-019-00706-z) contains supplementary such as Erwinia carotovora [8], Erwinia chrysanthemi material, which is available to authorized users. [9], Enterobacter aerogenes [10], Staphylococcus aureus [11], Thermus thermophilus [12], and Pseudomonas * Selma Allouache [email protected] aeruginosa [13]. L-asparaginase is used for the treatment of acute lymphoblastic leukemia and its efficiency has been especially dem- 1 Laboratoire de Microbiologie Appliquée LMA, Département de onstrated in children. However, the glutaminase activity asso- Biologie Physico-Chimique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, Targa ouzemour, Béjaïa, Algeria ciated to the asparaginase activity can trigger serious side effects, consisting mainly in coagulopathy, hepatitis, neurotox- 2 Laboratoire de Physico-Chimie des Matériaux et Catalyse LPCMC, Département de Physique, Faculté des Sciences Exactes, Université icity, and other pathologies. L-asparaginases devoid of gluta- de Bejaia, Targa ouzemour, Béjaïa, Algeria minase activity constitute thus attractive therapeutic candi- 3 Protein Chemistry Unit, Faculty of Medicine, Université Libre de dates. Interestingly, Erwinia carotovora asparaginase was de- Bruxelles, 808 Route de Lennik, 1070 Brussels, Belgium scribed to exhibit low glutaminase activity, therefore believed BioNanoSci. to have fewer side effects in the context of leukemia therapy 2.2 Transplanting Strains [14]. To date, commercial preparations of L-asparaginase We used Dickeya solani cultures, which is a pectinolytic have serious side effects giving the poor pharmacological Erwinia; indeed, the latter is formerly divided into two species profile of the chemotherapeutic agent. It is indeed neces- called Erwinia carotovora and Erwinia chrysanthemi. sary to have both a high dose and repeated administra- Because of changes in nomenclature due to taxonomic evolu- tions before obtaining the protective effect [15]. Thus, tion of these bacteria, E. carotovora became Pectobacterium there is an urgent requirement of L-asparaginase prepara- carotovorum and E. chrysanthemi is named Pectobacterium tions with improved efficacy, safety, and clinical proper- chrysanthemi [20]. The work of Samson et al. allowed the ties [16]. Nanotechnology constitutes an attractive way transfer of Pectobacterium chrysanthemi to Dickeya sp. [21]. because of its potential for achieving specific processes Dickeya solani used in the present work was isolated from and selectivity, especially in biological and pharmaceuti- potatoes in 2007 in the Netherlands (D. solani: IPO 2222, cal applications [17]. LMG 25993, NCPPB 4479; IPO: Plant Research

The titanium(IV) oxide (TiO2) has gained much attentions International, 6708 PB Wageningen Netherlands; due to owning a high refractive index, a good chemical stabil- BCCM/LMG: Belgian Coordinated Collection of ity, and an elevated dielectric constant in sensing applications Microorganisms/Laboratorium voor Microbiologie, [18]. It is used in paints, printing ink, rubber, paper, cosmetics, Universiteit Gent (UGent) K.L. Ledeganckstraat 35 B-9000 pharmaceuticals, sunscreens, car materials, implanted bioma- Gent, Belgium; NCPPB: The National Collection of Plant terials, and decomposing organic matters in wastewater, [16]. Pathogenic Bacteria, National Agri-Food Innovation It was reported that dual-core D-shaped SPR-based PCF with Campus, Sand Hutton, York, YO41 1LZ, UK [22]). These titanium as splitting barrier enhanced the performance of he- cultures were stored at room temperature in LPA medium moglobin sensing [19]. containing 3.0 g/L of yeast extract, 5.0 g/L of peptone, and Zinc oxide and L-asparaginase bioconjugate showed cyto- 15.0 g/L of agar. Nutrient broths are inoculated by colonies toxicity against MCF-7 cell line. Interestingly, cytotoxicity of from the strain and incubated during 12 h under steering at

TiO2 nanoparticles on cancer cell lines was also reported, 26.0 °C. These cultures are used to inoculate King B media at suggesting that this nanoparticle cytotoxicity, together with optimal growth temperatures of each strain for 24 h. L-asparaginase activity, may provide synergistic lethal effect against cancer cells [16]. 2.3 Preparation of Nutrient Broth and Bacterial In the present study, we aimed initially, and for the first Culture time, to verify whether bacterial strains of Dickeya solani were able to produce L-asparaginase(s). We then purified and char- The enzyme production was performed according to the pro- acterized the enzyme, while focusing on the glutaminase ac- tocol described by Kamble et al. [23], with some modifica- tivity. Furthermore, obtaining L-asparaginase with low gluta- tions. The isolated bacteria were grown in nutrient broth (Hi minase activity would be very interesting for its clinical appli- media Mv 088) containing 3.0 g/L of sterile meat extract, 5.0 cations. Enhancing L-asparaginase activity is also an interest- of g/L NaCl, and 5.0 g/L of peptone. Sterilization was per- ing clinical property. As a consequence, we aimed to assess formed at 121.0 °C for 15 min. A bacterial colony of each the influence of nanoparticles, e.g., TiO2 and ZnO, on the subspecies was selected in each Petri dish and transferred to enzymatic activity. 100.0 mL of nutrient broth under sterile conditions. The medium was incubated at 25.0 °C for 7 days. The pH of the culture medium was daily monitored and, if necessary, adjust- 2 Materials and Methods ed to 6.9 [18]. Another medium was prepared with the same composition but by adding 3.0 g/L of L-asparagine substrate. 2.1 Culture of Strains After a 7-day incubation period and optimal bacteria growth, the culture medium was filtered using Whatman paper No. 1 Before starting the culture, a gradual thawing from − 80 to to roughly remove insoluble material. The filtrate was then − 20 °C is required. A bacterial suspension was picked up subjected to ultracentrifugation (40,000×g, 20 min, 4.0 °C) with a sterile loop on a King B medium, at pH 6.5, con- to remove the bacterial mass. taining 20.0 g/L of peptone, 1.145 g/L of K2HPO4,1.5g/ LofMg2+, 15.0 g/L of agar, and 15 mL of glycerol, and 2.4 L-Asparaginase Activity Measurements then incubated at 26.0 °C for 24 h. A bacterial colony is inoculated on slant tubes containing LPA medium. These L-asparaginase catalyzes the L-asparagine to L-aspartic acid tubes are kept at room temperature and can be used for a and ammonia. The latter product reacts with the Nessler’s period not exceeding 6 months. reagent to produce a chromogenic product. The enzymatic BioNanoSci. assay mixture consisted of 900 μLofL-asparagine substrate 2.6 Purification of L-Asparaginase (100 mM) in Tris-HCl buffer (pH 8.6) and 100 μLofcrude bacterial extract. The reaction mixture was incubated at 37 °C The purification was done with two different culture media, for 30 min, and 100 μL of 15% trichloroacetic acid (TCA) with and without adding L-asparagine as a substrate (3.0 g/L). was added to stop the reaction. The reaction mixture was centrifuged at 10,000×g for 5 min to remove the insoluble 2.6.1 Purification of L-Asparaginase on HiTrap Q-Sepharose material, and the released ammonia was quantified colorimet- Fast Flow Coupled to AKTAprime System rically after the addition of 100 μLofNessler’s reagent to a sample containing 100 μL of supernatant and 800 μLofdis- The soluble crude extract was fractionated on a HiTrap Q- tilled water. The obtained mixture was vortexed and incubated Sepharose Fast Flow column (5.0 mL) coupled to an at room temperature for 10 min followed by absorbance mea- AKTAprime system at room temperature and 0.6 kPa. surements at 425 nm against a blank solution. The amount of Before loading the sample, the column was preequilibrated ammonia produced was determined using ammonium sulfate with 50 mM Tris-HCl buffer at pH 8.0 (5 column volumes). as a standard. One unit of enzyme activity (IU) was defined as The column was washed with the same buffer (10 column the amount of enzyme that liberates 1 μmol of ammonia per volumes) until the absorbance at 280 nm reached its baseline. minute at 37 °C. Specific activity is expressed as units per Bounded proteins were eluted with a linear gradient from 0 to milligram of protein [24]. The same protocol is followed to 1.0 M NaCl in 50.0 mM Tris-HCl buffer at pH 8.0 (20 column measure the enzymatic activity in the presence of ZnO and volumes) at a flow rate of 1.0 mL/mL, and fractions of 3.5 mL TiO2 nanoparticles. were collected. All the solutions are degassed and filtered through 0.22 μmbeforeuse. The resulting chromatographic fractions were analyzed by 2.5 Optimization of Bacterial Growth absorbance at 280 nm. Fractions showing L-asparaginase ac- and L-Asparaginase Production tivity (not shown) were pooled, before being concentrated up to the desired volume, by ultrafiltration (Amicon system, Five milliliters of the culture broth was withdrawn asep- membrane cutoff 3.0 kDa) and/or diafiltration (5000 tically from the flasks at an interval of every 24 h. The MWCO Vivaspin 15R concentrator). broth was filtered using Whatman filter paper No. 1 and then centrifuged (Rotina 380 R, Germany) at 9000g for 2.6.2 Further Purification of L-Asparaginase by FPLC-Gel 10 min. The supernatant thus obtained was used as crude Filtration on Superdex 75 pg Coupled to AKTAprime System extract for temperature and pH assessment of the L- asparaginase activity. To further improve the enzyme purity and homogeneity, L-asparaginase-rich fractions eluted from the HiTrap Q- Sepharose FF were pooled and dialyzed against water 2.5.1 Optimum Temperature Determination and then against 50.0 mM Tris-HCl buffer at pH 7.4 con- taining150.0mMNaClbeforetobesubmittedtoFPLC- Flasks (capacity (C) 250 mL, useful volume (UV) 100 mL) Superdex 75 pg, on an AKTAprime. The column (Hiload containing nutrient broth (pH 6.9) are seeded under the con- 16/600) was equilibrated and eluted with a 50.0 mM Tris- ditions indicated above. Optimum temperature was deter- HCl buffer at pH 7.4 containing 150.0 mM NaCl. mined both for bacterial growth through absorbance at 600- Running conditions were 1.0 mL/min, 3.0 mL/fraction, nm measurements and L-asparaginase activity assessment and 0.6 kPa. All the solutions were degassed and filtered [25]. through 0.22 μmbeforeuse.

2.7 SDS-PAGE Experiments 2.5.2 Optimum pH Determination The SDS-PAGE experiments were performed on precast From a preculture of 24 h (absorbance at 600 nm in between gels (ExcelGel, 245 × 110 × 0.5 mm, gradient 8–18%) 0.6 and 0.8) on nutrient broth at pH 6.9, flasks containing the using the Multiphor II kit from Amersham Biosciences, same medium (C 250 mL, UV 100 mL) at pH values ranging running conditions 600 V, 50 mA, and 35 W at constant from 5.5 to 9.0 are seeded and incubated for 7 days under temperature (15.0 ± 0.1 °C). The separation was towards continuous stirring at optimal growth temperature. Optimum the anode and bromophenol blue was used as the tracking pH for bacterial growth was followed by absorbance measure- dye. Staining was made with silver staining procedures ments at 600 nm, while L-asparaginase activity was carried out following manufacturer’s instructions. Molecular weight using L-asparagine as the substrate [25]. standards were hen egg white lysozyme (14.4 kDa), BioNanoSci. soybean trypsin inhibitor (21.5 kDa), carbonic anhydrase 3 Results (31.0 kDa), hen egg white ovalbumin (45.0 kDa), bovine serum albumin (66.2 kDa), and rabbit muscle phosphory- 3.1 Optimization of L-Asparaginase Growth lase b (97.4 kDa). and Production Parameters

The culture conditions (pH and temperature) are known to 2.8 L-Asparaginase Specificity Assay Towards affect the gene expression and the production of metabolites. L-Asparagine and L-Glutamine In addition, the selection of appropriate fermentation conditions is essential for both the microorganisms’ growth and the L-asparaginase substrate specificity was evaluated by testing production of their enzymes [29]. the two substrates, L-asparagine and L-glutamine; L-glutaminase activity was determined using L-glutamine as substrate 3.1.1 Optimum Temperature Determination according to Imada et al. A reaction mixture containing 0.5 mL of 0.04 M L-asparagine, 0.5 mL of 0.5 M in Tris- Figure 1 shows that the production of L-asparaginase, as evi- HCl buffer (pH 8.6), 0.5 mL of enzyme, and distilled water denced by its enzymatic activity, is also temperature- to a total volume of 2.0 mL was incubated at 37 °C for 30 min. dependent and follows the same trend as the growth curve, The reaction was stopped by adding 0.5 mL of 1.5 M trichlo- with the same optimum temperature at 35 °C. roacetic acid. Blank tubes were run by adding the enzyme preparation after the addition of trichloroacetic acid. To 3.1.2 Optimum pH Determination 3.7 mL of distilled water, 0.1 mL of the above mixtures and 0.2mLofNessler’s reagent were added. After keeping the We can see from Fig. 2 that Dickeya solani still grows in a mixture at 20 °C for 20 min, absorbance was measured at wide range of pH (from 5.5 to 9.0), with a maximum growth at 450 nm with a spectrophotometer (UVmini-1240 Shimadzu, pH 6.5. Japan). L-glutaminase activity was assayed following the Our results corroborate those reported by Sahu et al. show- same procedure except using L-glutamine instead of L-aspar- ing that both optimum growth of actinomycetes and maxi- agine [26]. mum L-asparaginase activity were observed in the pH range 7to8andat37°C[30]. An optimum pH and temperature of 2.9 Effect of TiO2 and ZnO Nanoparticles 7.4 and 37 °C, respectively, were obtained by Raha et al. for on L-Asparaginase Activity Cylindrocarpon obtusisporum MB-10 L-asparaginase [31]. Furthermore, Narayana et al. have reported a maximum enzy-

PS 90 commercial TiO2 NPs from Evonik Industries have maticactivityatpH7.0and35°CforaStreptomyces been used in our experiments. They are sold under the trade albidoflavus L-asparaginase [32]. mark AEROXIDE® TiO2 P 90 articles. They are a mixture of phases, 80% anatase and 20% rutile [27]. 3.2 Purification of L-Asparaginase Nanosized ZnO aerogel was synthesized by dissolving

16.0 g of zinc acetate dehydrate ((Zn(CH3COO)2,2H2O), After bacterial culture process, L-asparaginase was first 95%, Aldrich) in 112.0 mL ethanol. The mixture was kept isolated by using filtration, centrifugation, and before to under continuous magnetic stirring at room temperature dur- be further purified by chromatography techniques. The ing 20 min. To facilitate the complete dissolution of zinc ac- enzyme preparation obtained following the two chromato- etate in ethanol, a few drops of monoethanolamine (MEA) graphic techniques was used for the characterization were added to the solution. The homogeneous and transparent studies. solution obtained was placed in a 1-L capacity autoclave type The fractionation of the bacterial total soluble extract on the Parr 4848 Reactor for drying under supercritical conditions of cation-exchange support was presented on Fig. 3. ethanol (243 °C, 63 bar). To reach the critical temperature and As shown in Fig. 3, fractionation of the whole bacterial pressure with the used autoclave, a volume of 267.0 mL of soluble protein fraction on a HiTrap Q-Sepharose Fast Flow ethanol was added to the prepared solution. After cooling allowed to obtain four main peaks designated as pool I, pool II down to ambient temperature, a white powder was obtained. (both constituting the un-retained fraction), pool III, and pool A sample of this powder was annealed at 500 °C for 2 h in air IV. According to SDS-PAGE, only pool III contains proteins [28]. bands with apparent molecular weight of 33.0 kDa that may The L-asparaginase enzymatic activity in the presence of correspond to L-asparaginase. This fraction was submitted to

ZnO and TiO2 nanoparticles was performed following the size exclusion chromatography for further purification of the protocol described by Kamble et al. [23]. enzyme. As shown in Fig. 4, two less resolved minor peaks BioNanoSci.

Fig. 1 Optimum temperature of bacterial growth (circles) and L- asparaginase production (squares)

and a major peak were obtained. When subjected to SDS- confirming that the L-asparaginase production increases in the PAGE analysis, the major peak (line 2 in SDS-PAGE on the presence of this substrate. right side, pool IIIb in Fig. 4) reveals the presence of a nearly pure protein band migrating with an apparent molecular 3.3 Specificity Assessment of L-Asparaginase towards weight of 33 kDa. L-Asparagine Versus L-Glutamine When submitted to FPLC-Superdex 75 pg, the pools of peak III (PIII) of HiTrap Q-Sepharose Fast Flow was separat- When compared with the substrate L-asparagine, the rela- ed into two peaks, PIIIa (from fraction 6 to fraction 23) and tive enzymatic activity of Dickeya solani L-asparaginase −3 PIIIb (from fraction 24 to fraction 36) (Fig. 4). towards L-glutamine is 5.10 %. The purified L- It is interesting to note from Figs. 3 and 4 that the absor- asparaginase showed thus a high specificity against its bance at 280 nm, reflecting protein amount, is higher when the natural substrate L-asparagine. These findings are very bacterial culture medium is supplemented with L-asparagine, interesting and are 103 times lower than those reported

Fig. 2 Optimum pH of bacterial growth (circles) and L- asparaginase production (squares) BioNanoSci.

Fig. 3 Superposition of HiTrap Q-Sepharose Fast Flow chromatograms obtained in the presence (circles) and in the absence (squares) of L-asparagine in the bacterial culture medium

by El-Naggar et al. for L-asparaginase of Streptomyces The electrophoresis gel on the left side is a SDS-PAGE of brollosae NEAE-11 [33]. the peaks obtained after HiTrap Q-Sepharose (lines 1 and 2: pool I, lines 3 and 4: pool II, lines 5 and 6: pool III, and line 7: 3.4 SDS-PAGE Analysis molecular weight standards). However, the electrophoresis gel on the right side represents a SDS-PAGE of the peaks obtained after FPLC-Superdex 75 pg of pool III (line 1: pool IIIa and The results of SDS-PAGE analysis are summarized in line 2: pool IIIb). Fig. 5 which shows the electrophoretic results of the sam- ples passed on HiTrap Q-Sepharose and FPLC-Superdex 75 pg, respectively, the first gel allowed us to target the 3.5 Characterization of TiO2 and ZnO Nanoparticles pool which could contain our enzyme of interest which is pool III. The second gel confirmed the presence of L- The nanoparticle’s size was measured by XRD technique asparaginase on pool IIb. using the Rietveld analysis. The data showed a TiO2 diameter

Fig. 4 Superposition of FPLC- Superdex 75 pg chromatograms obtained in the presence (squares) and in the absence (circles) of L- asparagine in the culture medium BioNanoSci.

Fig. 5 SDS-PAGE of the peaks obtained after HiTrap Q- Sepharose Fast Flow and FPLC- Superdex 75 pg

of 13.5 ± 1.00 nm, which is in agreement with published char- 3.6 Effect of TiO2 and ZnO Nanoparticles acteristics of Evonik Industries. Such a product was developed on L-Asparaginase Activity to increase the photocatalytic properties of these nanoparticles. The obtained product is named AEROXIDE® TiO2 P As can be seen from Table 1, both the TiO2 and ZnO 90, which we used in this work. The product is characterized nanoparticles increased the activity of L-asparaginase. by an average size of 14 nm [27]. On the other hand, ZnO The results obtained however indicated a six-fold increase nanoparticles have an average diameter of (60.0 ± 10.0) nm. in the L-asparaginase specific activity in the presence of

The diffraction diagrams of TiO2 and ZnO nanoparticles are TiO2, while ZnO only induced a 2.5-fold specific activity presented in Figs. 6 and 7. enhancement. These results clearly show that TiO2

Fig. 6 Diffraction diagrams of TiO2 nanoparticles BioNanoSci.

Fig. 7 Diffraction diagrams of ZnO nanoparticles nanoparticles are more efficient than ZnO nanoparticles in calculated the intrinsic activity by dividing the measured improving L-asparaginase activity. However, this does not (specific) activity by the surface (square of the radius): exclude that some efficiency of ZnO nanoparticles will be ¼ = 2 ð Þ obtained by reducing their size. Intrinsic activity Specific activity r 1

In Eq. 1, we used the specific activity of the L-asparaginase 3.7 Comparison of the Specific Activity sample obtained after the chromatography on FPLC-Superdex of L-Asparaginase in the Presence of ZnO and TiO2 75 pg. The results were summarized in Table 1. Nanoparticles

Because of extreme small size of nanoparticles (13.5 nm for TiO2 and 60 nm for ZnO), most of their atoms are on the 4 Discussion surface. It is possible to determine by simple calculation which material is the most efficient for catalysis, e.g., by sep- Study of the effect of temperature on the growth of Dickeya arating the size effect from the catalytic effect of the material solani in the range of 20 to 45 °C showed a growth increase itself. To do this, and because the number of reaction centers is that reached a maximum at around 35 °C. At higher temper- proportional to the surface of the nanoparticles, we have ature, the bacterial growth decreased rapidly. Our data corroborate those of Janse and Ruissen [34]thatreportedanoptimal

Table 1 Effect of nanoparticle surface on the activity of purified L- growth temperature of Dickeya between 35 and 37 °C [12]. A asparaginase sharp increase in L-asparaginase production, as reflected by enzymatic activity measurements, was observed between 30 Nanoparticles TiO ZnO 2 and 35 °C. This may be due to low substrate transport through Nanoparticles size (nm) 14.0 60.0 cell membrane at low temperature [25, 32]. A rapid decrease Activity (IU/mL) 10.86 3.20 in L-asparaginase production was observed beyond 35 °C, Total activity (IU) 217.2 64.0 which may be explained by some thermal denaturation of Specific activity (IU) 108.6 32.0 the enzyme [35]. On the other hand, both the bacterial growth Intrinsic activity (AU) 0.550 0.008 and enzyme activity profiles superpose quite well and both of them are significantly affected by pH. The pH of the culture BioNanoSci. medium is indeed known to affect many enzymatic reactions, on a column of HiTrap Q-Sepharose Fast Flow and further e.g., by modifying the transport of some nutrients and en- purified on a FPLC-gel filtration chromatography on zymes through the cell membrane [36]. Although Dickeya Superdex 75. The purity of the L-asparaginase preparation solani still grows in a wide range of pH (5.5 to 9.0), it shows obtained from the gel filtration chromatography step was a maximum growth at pH 6.5. This observation is not surpris- assessed by SDS-PAGE. The results revealed the presence ing because the culture media generally used for growing of one protein band with an apparent molecular weight of Dickeya and for biochemical studies are quite often adjusted 33 kDa. Comparatively, an apparent molecular weight of to a pH in between 5.0 and 6.5 [37]. 42 kDa was reported for an L-asparaginase from Erwinia The L-amino acid asparagine is well known for being a carotovora [45], while an L-asparaginase of molecular weight significant factor to induce L-asparaginase production [25]. 32 kDa was isolated from Escherichia coli 055:B5 [46]. The The chromatograms obtained from ion-exchange chromatog- bibliographic data indicate that L-asparaginase is a raphy showed that addition of the substrate L-asparagine to homotetramer, which explains the occurrence of a single band culture medium induced L-asparaginase production compared in the electrophoretic profile on SDS-PAGE [47, 48]. The with the medium freed of this substrate. This was clearly functional L-asparaginase from E. coli and Erwinia is a reflected by the enhancement of the absorbance at 280 nm homotetramer [36]. Some authors who have worked on of the extract obtained in the presence of L-asparagine. Erwinia L-asparaginase have reported that the molecular These results were in agreement with those reported in the weight of L-asparaginase subunit is between 32.5 and literature [25, 38, 39], underlying thus the importance of L- 38 kDa [49], and others report a value of 34.5 kDa [50]. asparagine in improving L-asparaginase production. Mahajan et al. reported that the L-asparaginase produced by Substrate specificity studies indicated that L-asparaginase Bacillus licheniformis was found to be a homotetramer protein has greater affinity towards L-asparagine compared with L- of 134.8 kDa, with monomeric size of 33.7 kDa [51]. glutamine. Compared with L-asparagine, the relative activity It is known that nanoparticles can have an activating effect of Dickeya solani L-asparaginase towards L-glutamine is on some enzymes. It has been shown for example that silver −3 5.10 %. This very low L-glutaminase activity is very inter- and gold nanoparticles have activating effects on neurotrans- esting when the enzyme is designed for therapeutic purposes, mitters in the case of monoamino oxidase and choline esterase such as for the treatment of leukemia, allowing the reduction [52]. From our results it can be concluded also that both TiO2 of side effects. The use of L-asparaginase as a therapeutic and ZnO nanoparticles increase the activity of L-asparaginase. agent is principally hampered by the side effects that it can In some cases, however, nanoparticles can decrease the cause [40], such us hyperglycemia, hemorrhagic pancreatitis, enzymatic activity, as was reported for horseradish peroxidase neurotoxicity [41], and immunosuppression [30]. (HRP) using 3,3′,5,5′-tetramethylbenzidine (TMB) as sub- Table 2 shows the decrease in the total amount of proteins strate. The results indicate that while the gold nanoparticles and the increase in the L-asparaginase specific activity. The decreased the enzymatic activity, the silver nanoparticles en- purification of L-asparaginase was carried out by two chro- hanced it [53]. It has also been shown that some nanoparticles matographic steps as shown in Table 2, with a final yield of properties, such as size, shape, surface chemistry, and charge 14.4% and a purification fold of 81.8. L-asparaginase from can alter the structure and function of enzymes [54]. Besides, Streptomyces albidoflavus which has been purified using it has been demonstrated that nanoparticles are able to induce CM-Sephadex C-50 column up to 83.0-fold [43]. Amenas protein modifications [55]. et al. have reported a 85.0 purification fold and a yield of We clearly demonstrated that TiO2 nanoparticles induced 32% of an L-asparaginase from Streptomyces sp. PDK2 by an enhancement of L-asparaginase activity, which could be using Sephadex G-200 gel filtration as a final purification step exploited to improve the therapeutic efficiency of this enzyme. [44]. Enhancing L-asparaginase activity is a prerequisite to avoid In the present study, Dickeya solani L-asparaginase purifi- secondary effects that are linked, e.g., to high dose and repeat- cation was achieved using anion-exchange chromatography ed administration of this drug.

Table 2 Summary of the purification steps of L-asparaginase from Dickeya solani. Protein quantification was carried out using the Bradford method, using BSA as a standard protein [42]

Step Volume (mL) Protein (mg) Activity (IU/mL) Total activity (IU) Specific activity (IU/mg) Yield (%) Purification fold

Crude extract 100 10.9 2.5 250 0.22 100 – HiTrap Q-Sepharose FF 40 0.82 2.2 88 2.68 35.2 12.1 FPLC-Superdex 75 pg 20 0.1 1.8 36 18.0 14.4 81.8 BioNanoSci.

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Bioscience, Biotechnology, and icochemical properties and optimal concentration of nanopar- Biochemistry, 59,749–750. ticles to try to improve the specific activity of L-asparaginase. 11. Muley, R. G., Sarker, S., Ambedkar, S., & Nail, S. R. (1998). Influence of alkali treated corn steep liquor containing medium on Our results also revealed that L-asparaginase isolated from the protein production by Staphylococcus aureus. Folia Microbiology., bacterial strains Dickeya solani showed high L-asparaginase 43,31–34. specificity, highlighting its potential use as a therapeutic agent, 12. Prista, A. A., & Kyridio, D. A. (2001). L-asparaginase of essentially because of the absence of L-glutaminase activity. It thermothermophilus: purification, properties and identification of would be thus interesting to evaluate its potentialities as an essential amino acids for catalytic activity. 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