Puri cation and immobilization of L-asparaginase from Citrobacter freundii EGY-NE1

Mohamed Mousa (  mousa1451967@gmail.com ) Damietta University Faculty of Science Mahmoud M. Nour El-Dein Botany and Microbiology Department, Faculty of Science, Damietta University, New Damietta, Egypt Mohamed I. Abou-Dobara Botany and Microbiology Department, Faculty of Science, Damietta University, New Damietta, Egypt Nashwa E. Metwally Botany and Microbiology Department, Faculty of Science, Damietta University, New Damietta, Egypt


Introduction
L-asparaginase (L-asparagine amidohydrolase, E.C.3.5.1.1) is widely used in the clinical and food industries for converting L-asparagine to aspartate. As a food additive, it reduces the risk of acrylamide formation (Pedreschi et al. 2008). As an antileukemic drug, it consumes asparagine; hence, kills tumor cells (Benchamin and Roshan 2019). It is also used as a biosensor of asparagine in leukemia and food industries (Verma et al. 2012). For these applications, researchers explore L-asparaginases from several microorganisms (Izadpanah Qeshmi et al. 2014).
Bacterial L-asparaginases are classi ed into two types; L-asparaginase I is constitutively secreted intracellular and L-asparaginase II is extracellularly secreted in response to nitrogen starvation (Izadpanah et al. 2018). L-asparaginase II represents the applicable enzyme because it has more a nity than type I for L-asparagine (Castro et al. 2021) and accumulates in broth cultures, which facilitating downstream processing (Amena et al. 2010).
The downstream processing of the enzyme includes its puri cation and immobilization. The pure enzyme allows studying its molecular, physiological and Kinetic properties (Nadeem et al. 2009(Nadeem et al. , 2015. Different immobilization methods (including adsorption onto solid carriers, entrapment in polymeric gels, encapsulation in membranes, cross-linking with bifunctional or multifunctional reagents and linking to an insoluble carrier (Klibanov 1983)) limits the mobility of the enzyme, so it facilitates the control of the reaction endpoint and increases the operational stability (Robert DiCosimo et al. 2013; Ahmad and Sardar Although several commercial L-asparaginases are available (Batool et al. 2016), industries still require stable L-asparaginases possessing a high a nity to asparagine (Chand et al. 2020). Since 1966, type II Lasparaginase from E. coli was clinically used as an anticancer drug (Borek and Jaskólski 2001). However, it causes hypersensitivity side effects (Pochedly 1977) due to its glutaminase activity against normal cells (Howard and Carpenter 1972). In 2011, FDA approved Erwinia chrysanthemi-derived asparaginase for patients with hypersensitivity to E. coli-derived asparaginase (Salzer et al. 2014); nevertheless, this is also limited by clinical side effects (Moola et al. 1994).
Thus, researchers still explore and characterize L-asparaginases from new sources to reduce production costs and avoid clinical side effects. Citrobacter represents a potential new source of L-asparaginases that was rarely studied; to our knowledge, only intracellular rather than extracellular L-asparaginases from Citrobacter were only twice studied (Bascomb et al. 1975;Davidson et al. 1977). This study aims to purify and characterize extracellular type II L-asparaginases from Citrobacter freundii EGY-NE1; then, immobilize it via encapsulation in Ca-alginate to improve its stability and reusability.

Materials And Methods
The bacterial strain Citrobacter freundii EGY-NE1 was isolated from Egyptian clay soil (Damietta) and identi ed according to its phenotypic and molecular characters (Frederiksen 2005) by Rizk (2019).

Growth medium and conditions
Citrobacter freundii EGY-NE1 was cultured on tryptone-glucose-yeast extract broth medium (tryptone 5.0 g, yeast extract 5.0 g, asparagine 10 g, glucose 1.0 g, K 2 HPO 4 1.0 g; pH 6.0) at 37 °C and 75 rpm for 48 hours. The supernatants of grown cultures were separated by centrifugation at 7,000 rpm for 10 minutes.

L-asparaginase assay
We determined the L-asparaginase activity by assaying the released ammonia during the hydrolysis reaction of asparagine by the enzyme extracts (Prakasham et al. 2009). A mixture of the enzyme extract (0.1 ml), Tris-HCl buffer (0.2 ml, 0.05M, pH 8.6) and L-asparagine solution (1.7 ml, 0.01M) was incubated for 10 minutes at 37°C. The reaction was stopped by adding 0.5 ml of 1.5 M trichloroacetic acid followed by centrifugation at 1000 rpm; 0.5 ml of the supernatant was diluted to 7 ml with distilled water and 1 ml of Nessler's reagent was added. After 10 minutes, the absorbance of the developed color was measured at 480 nm by a spectrophotometer (model UV1100, Shanghai Yoke Instrument Company, China). One unit of L-asparaginase was de ned as the amount of enzyme that liberates one µmole of ammonia per minute (Prakasham et al. 2009).

Protein Estimation
Total protein was estimated according to the method of Bradford (1976).
Puri cation of L-asparaginase from Citrobacter freundii Proteins in the crude enzyme preparations were precipitated under cold conditions by using different concentrations of ammonium sulfate (40,50,60,70, and 80%) with stirring for 20 minutes; then, we left it overnight at 4°C for complete precipitation. The precipitated proteins were separated at 5000 rpm for 20 minutes then suspended in sodium phosphate buffer (0.1M, pH 7). These enzyme preparations were put in dialysis tubes against 0.05 M sodium phosphate buffer (pH 7) overnight at 4˚C to get rid of salts.
Gel ltration chromatography followed by anion exchange chromatography was used to purify the enzyme. The enzyme preparation was applied to a gel ltration Sephadex G-50 column (55 × 1.5 cm) equilibrated by Tris-HCl buffer (0.05 M, pH 8), then eluted by the same buffer. The eluted fractions (2 ml) were assayed for L-asparaginase activity and protein content. Active fractions were pooled for further puri cation by anion exchange chromatography.
The active fractions were applied to an anion exchange column of DEAE-cellulose (30 × 1.5 cm) equilibrated with Tris-HCl buffer (0.05 M, pH 8). The proteins were eluted with a linear gradient of 0.0 to 1.0 M NaCl in the same buffer; each fraction (2 ml) was assayed for L-asparaginase activity and protein content.
Sodium dodecyl sulfate poly-acrylamide gel electrophoresis Sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) was used for qualifying and comparing protein samples according to the method of Laemmli (1970). Protein samples were boiled for 2 minutes in the sample-loading buffer. The molecular weight of protein bands was referenced to a prestained protein marker (# SM 0671, Fermentas). Electrophoresis was carried out by Bio-Rad Mini-Protein II cell gel apparatus at 120 volts.
The separated proteins on the gel were stained in Coomassie brilliant blue R-250 for one hour; then, soaked in a de-staining solution (H 2 O: methanol: acetic acid 50:40:10) for two hours until the protein bands appeared.
Activity staining of L-asparaginase Activity staining was performed in a Petri dish containing L-asparagine gel (1% L-asparagine, 1% agar dissolved in 25 ml of 0.05 M Tris-HCl buffer; pH 8.0). We added the enzyme solution (20 μl) to a formed well in the gel; then, incubated it at 37°C. After 30 minutes, the gel was stained with Nessler's reagent; the formation of a brown zone indicates L-asparaginase activity (Lincoln et al. 2019).
Biochemical and kinetic properties of the puri ed L-asparaginase We studied the enzyme activity at various temperatures by incubating the assay reaction mixtures at different temperatures (31,34,37,40,43 and 46 °C). To study the effect of pH on L-asparaginase activity, we adjusted pH of Tris-HCl buffer (the standard buffer of the assay) to different pH values (3, 4, 5, 6, 7, 8, 9 and 10). The activities were expressed as a relative percent to the maximal activity.
The effects of metal salts (NaCl, KCl, CaCl 2 , ZnCl 2 , BaCl 2 , MgCl 2 and CaSO 4 ) and inhibitors (sodium azide, sodium tartrate, mercuric chloride, EDTA and sodium EDTA) on enzymatic activity were investigated. The puri ed enzyme (0.2 ml) was mixed with the salt or inhibitor solutions at 10 mM nal concentration; then, incubated at room temperature for 30 minutes before the enzyme assay. The activities were expressed as a relative percent to the control.
For the kinetic properties, the enzyme activity was assayed by using reaction mixtures containing different L-asparagine concentrations (9,10,20,30,40,50 and 60 mM). The Km and Vmax values were calculated from Lineweaver-Burk plots.
Immobilization of the crude and puri ed L-asparaginase by encapsulation in Ca-alginate The enzyme was encapsulated in Ca-alginate beads as described by Ulu and Ates (2017). We mixed the crude or puri ed L-asparaginase (10 ml) with Na-alginate solution (40 ml, 2%); this mixture was extruded (from 3 cm controlled distance) in 2% CaCl 2 solution using a micro-pipette. After 30 minutes, the formed beads were ltrated; then, washed with a sterile NaCl solution (0.9%) followed by sterile distilled water (three times). The protein content and enzymes activity were assayed after immobilization.

Characterization of the immobilized enzymes morphology
Morphology of immobilized crude and puri ed L-asparaginase was examined by scanning electron microscope (SEM, JEOL, JSM IT200, Japan).
Effect of temperature and pH on the immobilized crude and puri ed L-asparaginase To determine the effect of temperature on the immobilized crude and puri ed L-asparaginase, the assay reaction mixture was incubated at different temperatures (31,34,37,40,43 and 46 °C). The effect of different pH was also studied by adjusting pH of the standard assay buffer to different values (3, 4, 5, 6, 7, 8, 9 and 10). The thermal stability of the immobilized crude and puri ed L-asparaginase was studied at 40˚C. The enzyme beads were incubated at 40˚C for 60 miutes, without a substrate; the enzyme activity was assayed at 10 minutes intervals. The activities of the enzyme were expressed as a percent of the maximal activity.

Reusability of the immobilized enzyme
The reusability of the immobilized crude and puri ed enzyme were tested up to 5 rounds; the enzyme beads were incubated with the reaction mixture at optimum conditions for the assay; at the end of the reaction, the beads were removed from the reaction mixture and washed with distilled water; these beads were repeatedly used in the subsequent four assay rounds. The activities of the enzyme were expressed as a percent of its initial activity.

Results
Puri cation of L-asparaginase from Citrobacter freundii Extracellular L-asparaginase from Citrobacter freundii was precipitated using ammonium sulfate followed by puri cation by chromatographic techniques. The optimum ammonium sulfate concentration for L-asparaginase precipitation was 50-60% (Table 1). The precipitated enzyme was then dialyzed and concentrated on a sucrose bead. When the concentrated enzyme was applied to the sephadex G-50 column, three large active peaks were detected (Figure 1). L-asparaginase speci c activity (in these active peaks) increased from 28.12 U/mg protein to 37.73 U/mg protein; that step recovered 26.28% of the activity and puri ed the enzyme by 3.75 fold ( Table 2).
The active fractions obtained from gel ltration were further puri ed using anion exchange chromatography (DEAE-cellulose column); active fractions of the large peak ( Figure 2) showed speci c enzyme activity of 98.617 U/mg proteins; this nally puri ed the enzyme to 5.83 fold and recovered 25.76% of L-asparaginase from Citrobacter freundii ( Table 2).
SDS-PAGE of the puri ed L-asparaginase Figure 3 shows the protein pro les of the crude and the puri ed enzyme by Sephadex G-50 and DEAEcellulose columns. Two protein bands of approximate molecular weight 19 kDa were detected in the pure enzyme sample.

Activity staining of L-asparaginase
The L-asparaginase activity of the puri ed protein was con rmed by the active staining; a brownishyellow spot (indicating L-asparagine degradation by L-asparaginase) was developed only in the puri ed and crude sample (Figure 4).
Biochemical and kinetic properties of the puri ed L-asparaginase The enzyme exhibited its maximal activity at 37 °C ( Figure 5). While, the activity declined at temperatures below or above this optimal value; the enzyme relative activities were 48.73 % and 54.05 % at 34 °C and 40 °C, respectively. The enzyme activity peaked at pH 8 ( Figure 6); however, the enzyme retained only 75.27 % and 53.86 % of its activity at pH 7 and pH 9, respectively. The enzyme kept more than 50 % of its activity through the range from pH 5 to pH 9.
The activity of the puri ed enzyme increased gradually with increasing L-asparagine concentration until saturation at 40 mM. The lineweaver-Burk plot (Figure 7) shows the a nity of the enzyme to Lasparagine. The K m and V max of the puri ed enzyme were 0.0179 M and 2.66 U/ml, respectively.
The cations Ca 2+ , Mg 2+ , K + and Ba +2 activated L-asparaginase from C. freundii. The Zn +2 cation slightly inhibited the enzyme, while Na + moderately inhibited it (Table 3). EDTA and mercuric chloride represented the strongest inhibitors among the tested inhibitors (Table 3). Table 3 also shows the moderate inhibition of azide and tartrate to the enzyme.

Characterization of the immobilized crude and puri ed enzyme
Morphology of the entrapped L-asparaginase on Ca-alginate was examined by scanning electron microscope; the diameters of the entrapped proteins ranged from 42.17 to 47.37 nm and from 46.78 to 71.97 nm for the immobilized crude and puri ed enzymes, respectively (Figure 8.).
At the temperature range from 31 °C to 43 °C, both the crude and puri ed immobilized L-asparaginase retained more than 60 % of their activity ( Figure. 9). The crude exhibited its maximal activity at 34 °C to 37 °C, while the puri ed exhibited its maximal activity only at 37 °C; the relative activity of the puri ed immobilized enzyme at 34 °C was 87 %. The crude and puri ed immobilized L-asparaginase kept their maximal activity for 10 minutes at 40 ˚C ( Figure. 10); after 60 minutes, they retained 76 % and 61 % of their activity, respectively. Figure 11 shows that the activities of the crude and puri ed immobilized L-asparaginase increased gradually over a pH range of 3.0 to 8.0 (an optimum pH of 8.0); above pH 8 to pH 10, the activities slightly declined.
Regarding the reusability of the enzyme, both the immobilized enzymes kept their activities over the tested 5 cycles (Figure. 12); at the end of the 5 th cycle, the immobilized puri ed and crude enzymes kept 91% and 89 % of their initial activities, respectively. Discussion L-asparaginase from C. freundii EGY-NE1 represents a promising candidate for biotechnological applications; its puri cation is the rst step for its downstream processing. The puri cation approach used by this research (precipitation by ammonium sulfate followed by chromatographic techniques) was adopted by several studies (Batool et al. 2016); the precipitation step is usually followed by dialysis to remove excess salts (Makky et al. 2014). In comparing with those studies, our puri cation process  ( Scheetz et al. 1971). But, few L-asparaginases exhibited low molecular weights below 20 kDa; L-asparaginase from Streptobacillus sp. KK2S4 (Makky et al. 2014) and Flammulina velutipes ( Eisele et al. 2011) were reported at 11.2 and 13 kDa, respectively.
The optimum temperature of the puri ed L-asparaginase from C. freundii EGY-NE1 (37°C) is the same human body temperature; this allows elimination of the body-asparagine upon in vivo treatment (Elshafei 2012); this also increases the half-life time of the enzyme in the serum; hence, avoid the need to multipledose causing hypersensitivity (Krishnapura et al. 2015). L-asparaginase's optimum temperatures differ from one species to another; however, those often range between 25°C and 45°C (Chand et al. 2020 Metal ions interface with proteins as electron donors or acceptors (Buchholz et al. 2012); hence, they can inhibit or activate enzymes by regulating the multimeric structure of the enzyme or the enzyme reaction intermediates (Krishnapura et al. 2015). However, the same metal ion or metal chelator can differently in uence the L-asparaginases from different sources. While Ca 2+ , Mg 2+ , K + and Ba 2+ activated Lasparaginase from C. freundii EGY-NE1, all these ions except K + inhibited L-asparaginase from Bacillus aryabhattai ITHBHU02 (Singh et al. 2013); Mg 2+ also inhibited L-asparaginase from Cladosporium sp. (Mohan Kumar and Manonmani 2012). In contrast, Na + inhibited L-asparaginase from C. freundii EGY-NE1and activated that enzyme from Bacillus aryabhattai (Singh et al. 2013). L-asparaginase from C. freundii EGY-NE1, similar to that from Pseudomonas stutzeri MB-405 (Manna et al. 1995), was moderately inhibited by Na + , Hg 2+ Zn 2+ and EDTA. Thus, data from the inhibition study help to improve the enzyme catalytic e ciency (Krishnapura et al. 2015); for L-asparaginase from C. freundii EGY-NE1, the presence of Ca 2+ , Mg 2+ , K + or Ba 2+ improves the enzyme catalytic e ciency.
Immobilization of L-asparaginases can enhance their thermal and pH stabilities in addition to their selectivity (Nunes et al. 2020); several immobilization methods and carriers have been developed. In this study, L-asparaginase was entrapped in Ca alginate beads; this generally enhanced the enzyme thermal stability and pH range. The properties of immobilized enzymes are governed by the characters of the enzyme, the carrier material and the speci c interaction of the immobilization method. Immobilization of the recombinant L-asparaginase from Erwinia chrysanthemi 3937 on epoxy activated Sepharose CL-6B enhanced its stability it at 4°C (Kotzia and Labrou 2011). The L-asparaginase fatty acid bio-conjugates improved the kinetic properties, the biological half-life, hence, the in-vivo antitumor activity of the immobilized L-asparaginase (Ashra et al. 2013). We rstly con rmed the e ciency of entrapment of Lasparaginase from C. freundii EGY-NE1 (both crude and puri ed enzymes) on Ca-alginate by examining their morphology by SEM; then we characterized their temperature and pH ranges.
The immobilized L-asparaginase from C. freundii EGY-NE1 kept the same optimum temperature (37 ˚C) of the free puri ed enzyme. But, the immobilized enzyme retained more activity than the free one at different temperatures along the tested range; this may be attributed to protection of the tertiary structure of the enzyme by the support material (Martinek et al. 1977). The immobilization of L-asparaginase from C. freundii EGY-NE1 on Ca alginate also increased the enzyme half life time at 40 ˚C; this is an expected advantage since immobilization by entrapment method protects enzymes against thermal denaturation by increasing their rigidity (Abdel-Naby 1993; Chang and Juang 2005).
The optimum pH of the immobilized L-asparaginase from C. freundii EGY-NE1 was pH 8.0, the same for the free enzyme. As a comparison, the immobilized L-asparaginase retained higher activities than the free puri ed enzyme along the pH range; both the immobilized puri ed and free puri ed L-asparaginase recorded their minimum relative activities at pH 3 as 75 % and 34.11 %, respectively. The solid matrix may modify the pH in the enzyme micro-environment, as compared to the bulk environment, through its surface and residual charges (Abdel-Naby 1993). Interactions between the enzyme and its carrier may also affect the intra-molecular forces maintaining the enzyme conformation, which would affect the enzyme activity (Talekar et al. 2010).
The enzyme immobilization guarantees the reusability of enzyme preparation. Thus, it permits a low cost for industrial use. Immobilization of L-asparaginase from C. freundii EGY-NE1 on Ca alginate beads would be useful for its application; these beads can be further packed in a column to hydrolyze asparagine. However, repeated reactions may reduce the enzyme's catalytic e ciency by weakening its binding to the carrier or distorting its active site (Abdel-Naby 1993). The immobilized L-asparaginase, in this study, kept 91 % of its catalytic e ciency even after the 5th cycle of reusability.

Conclusions
This study offers a promising new microbial L-asparaginase for applications. This extracellular type II Lasparaginase from Citrobacter freundii EGY-NE1 possess a low molecular weight, and well activity at temperature and pH of the human body; further in-vivo studies are needed to con rm its e cacy and safety as an anticancer agent for clinical application. The enzyme encapsulation in Ca-alginate beads improved its stability and reusability allowing an economic application in industries.
Abbreviations SDS-PAGE : Sodium dodecyl sulfate poly-acrylamide gel electrophoresis Declarations Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author upon request.

Competing interests
The authors declare that they have no competing interests.   Tables   Table 1. Activities of L-asparaginase from Citrobacter freundii in the supernatants and precipitates during its precipitation by different concentrations of ammonium sulfate. column (Sephadex G-50) and anion exchange column (DEAE-cellulose). Table 3. Effect of metal salts and inhibitors on the activity of the puri ed L-asparaginase from Citrobacter freundii. The enzyme activities are expressed relative to a control activity.  Figure 1 Puri cation of L-asparaginase from Citrobacter freundii using a gel ltration column of sephadex G-50.

Figure 2
Puri cation of L-asparaginase from Citrobacter freundii using an anion exchange column of DEAEcellulose.