Biochemical Characterization of <TEX>$\small{L}$</TEX>-Asparaginase in NaCl-Tolerant Staphylococcus sp. OJ82 Isolated from Fermented Seafood
Biochemical Characterization of $\small{L}$-Asparaginase in NaCl-Tolerant Staphylococcus sp. OJ82 Isolated from Fermented Seafood
Journal of Microbiology and Biotechnology. 2014. Aug, 24(8): 1096-1104
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : May 09, 2014
  • Accepted : May 26, 2014
  • Published : August 28, 2014
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Sangwon, Han
Jaejoon, Jung
Woojun, Park

L -Asparaginase from gram-positive bacteria has been poorly explored. We conducted recombinant overexpression and purification of L -asparaginase from Staphylococcus sp. OJ82 (SoAsn) isolated from Korean fermented seafood to evaluate its biotechnological potential as an antileukemic agent. SoAsn was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of 37.5 kDa, determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with asparaginases in gram-negative bacteria, size-exclusion chromatography determined SoAsn as a homodimer. Interestingly, the optimal temperature of SoAsn was 37℃ and over 90% of activity was retained between 37℃ and 50℃, and its thermal stability range was narrower than that of commercial E. coli L -asparaginase (EcAsn). Both SoAsn and EcAsn were active between pH 9 and 10, although their overall pH-dependent enzyme activities were slightly different. The K m value of SoAsn was 2.2 mM, which is higher than that of EcAsn. Among eight metals tested for enzyme activity, cobalt and magnesium greatly enhanced the SoAsn and EcAsn activity, respectively. Interestingly, SoAsn retained more than 60% of its activity under 2 M NaCl condition, but the activity of EcAsn was reduced to 48%. Overall, the biochemical characteristics of SoAsn were similar to those of EcAsn, but its kinetics, cofactor requirements, and NaCl tolerance differed from those of EcAsn.
L -Asparaginase (E.C. belongs to the amidohydrolase family, which converts asparagine to aspartic acid and ammonia [4 , 5] . The presence of L -asparaginase in guineapig serum was first discovered in 1922 and the antitumor properties of the enzyme were recognized later in 1961 [7 , 19] . Consequently, many studies showed that the amino acid L -asparagine is an absolute nutrient for mouse tumor cells [12 , 17 , 30] . Although L -asparagine is an essential amino acid for the growth of tumor cells, it is not required for the growth of normal cells because they can synthesize sufficient amount for their metabolic needs using the enzyme L -asparagine synthetase [9] . The presence of L -asparaginase deprives tumor cells of exogeneous L -asparagine and the suppressed growth of tumor cell leads to chemotherapy. Thus, the study of this enzyme could be crucial for development of a potent antitumor or antileukemic drug [5] . Another application of L -asparaginase is found in the baking process where the Maillard reaction typically occurs at temperatures above 120℃, resulting not only in a brown color and roasted flavor but also undesirable carcinogenic by-products. Acrylamide is a carcinogenic compound resulting from the reaction between sugar and the amino acid asparagine above 180℃ [7 , 10] . L -Asparaginase treatment to flour reduces acrylamide production; thus, to improve food safety, commercial products such as PreventASe (DSM, The Netherland) and Acrylaway (Novozymes, Denmark) produced from Aspergillus niger and Aspergillus oryzae , respectively, have been developed [10 , 28] . In food industry applications, fungal and bacterial sources of L -asparaginase dominate because plant- and animal-based enzyme sources produce insufficient quantities. L -Asparaginase purified from bacterial sources, including Escherichia coli , Erwinia chrysanthemi , and Serratia marcescens , are used in these applications [1 , 15 , 16 , 22 , 25 , 26 , 32] .
There are two types of L -asparaginase. Type II L -asparaginase exists in the periplasmic region, whereas type I L -asparaginase is located in the cytosol. Compared with type I L -asparaginase, type II L -asparaginase has a higher substrate affinity, thereby, it is more efficient to selectively degrade L -asparagine rather than L -glutamine. Most studies have investigated type II L -asparaginases that are preferred for clinical and industrial application [18 , 26] . Previous studies have investigated L -asparaginase from animal, plant, fungal, and bacterial sources; however, approved applications have been limited to a few species [11 , 18] . Biochemical characterizations of L -asparaginase from alternative sources have been conducted because of the immune-toxic side effects of the enzymes produced by E. coli and Erwinia carotovora [12] . Moreover, recent studies have undertaken structure-based trials to alter the amino acid sequences of E. coli L -asparaginase (EcAsn) to minimize its immunogenicity [12] . Other studies have shown that varying the growth media compositions and culture conditions can improve L -asparaginase production [1 , 17 , 22 , 25 , 26] .
This study targeted the recently isolated salt-tolerant species Staphylococcus sp. OJ82 as a novel L -asparaginase source [27] . In-depth studies of gram-positive species as new sources of L -asparaginase are worthwhile, because to date, Bacillus subtilis is the sole gram-positive source of this enzyme; other microbial sources are gram-negative bacteria. The aim of this study was to facilitate the molecular cloning, purification, and characterization of recombinant L -asparaginase from Staphylococcus sp. OJ82 (SoAsn). In particular, we tried to verify that SoAsn was active under extreme NaCl conditions (up to 25% (w/v)). Moreover, we determined the structural properties and biochemical characteristics of SoAsn.
Materials and Methods
- Bacterial Strains, Plasmids, and Culture Conditions
In a previous study, we isolated Staphylococcus sp. OJ82 from the traditional Korean fermented squid seafood ojingeo-jeotgal [27] . Staphylococcus sp. OJ82 was deposited in the Korean Agricultural Culture Collection (KACC) under the accession number KACC16993. Staphylococcus sp. OJ82 cells were incubated at 30℃ in Luria-Bertani (LB) medium with aeration via shaking at 220 rpm. pET-28a-c(+) (Novagen, USA), which contains a histidine tagging site, was selected for cloning. Competent cells from E. coli TOP10 were used for plasmid proliferation, and E. coli BL21 (DE3) was used as the L -asparaginase-expressing strain. E. coli TOP10 and E. coli BL21 (DE3) h arboring pET-28a-c(+) were g rown in LB m edium containing kanamycin (50 μg/ml) at 37℃.
- Reagents
To compare the biochemical properties of SoAsn and commercial L -asparaginase from microbial sources, we purchased lyophilized EcAsn (Sigma Aldrich, USA). The medium components were bacteriological-grade Bio-tryptone, NaCl (Bioshop, Canada), and yeast extract (M-biotech, Korea). Metal reagents were KCl, FeCl 2 (Junsei, Japan), MgSO 4 , CaCl 2 , CoCl 2 (S.P.C.GR Reagent, Japan), NiCl 2 (Shinyo Pure Chemicals, Japan), ZnCl 2 , L -asparagine, and L -glutamine (Sigma Aldrich).
- Cloning of the Overexpression Strain
Primers SOJ_15970 forward (5’-CGC GAATTC ATGAAA AAA CTTCTTCTC-3’) and SOJ_15970 reverse (5’-CGC- CTCGAG -CCA CGGCGGTATTATCACTCAAA-3’) were used to amplify SOJ_15970 encoding for L -asparaginase. The underlined nucleotides are the restriction sites for Eco RI and Xho I (Thermo Scientific, USA), respectively. Conditions for polymerase chain reaction (PCR) were at 94℃ for 90 sec, 35 cycles of denaturation at 94℃ for 45 sec, annealing at 58℃ for 45 sec, and polymerization at 72℃ for 45 sec, followed by final extention at 72℃ for 5 min. SOJ_15970 and pET-28a-c(+) were digested with Eco RI and Xho I and ligated by T4 ligase. Ligated product was transformed into E. coli TOP 10 and confirmed by PCR with the T7 promoter universal primer (5’-TAATACGACTACTCACTATAGGG-3’) and SOJ_15970 reverse primer. We confirmed the sequence of inserted SOJ_15970 was identical to the naïve SOJ_15970 of OJ82. pET-28a-c(+)-SOJ_15970 was transformed into E. coli BL21 (DE3) via electroporation and confirmed by PCR with the T7 universal promoter primer and SOJ_15970 reverse primer.
- Enzyme Expression and Purification
IsopropyL-β- D -thiogalactopyranoside (IPTG) was added to exponentially growing E. coli BL21 (pET-28a-c(+)-SOJ_15970) to induce the inserted SOJ_15970. After 4 h, cells were harvested by centrifugation and washed twice with phosphate-buffered saline. The cells were suspended in buffer (30 mM imidazole, 500 mM NaCl, 20 mM Na 2 HPO 4 ) and subsequently sonicated. Purification was carried out with affinity chromatography using a Hi-Trap FF nickel nitrilotriacetic acid (Ni-NTA) column (GE Healthcare Life Science, UK) connected to an AKTA FPLC Purifier 10 (GE Healthcare Life Science, UK) equilibrated with equilibration buffer (30 mM imidazole, 500 mM NaCl, 20 mM Na 2 HPO 4 ). SoAsn was concentrated using an Amicon Ultra 4 centrifugal filter (Millipore, Ireland) with Tris buffer (50 mM, pH 8.6). The protein concentration of the purified enzyme was measured using the Bradford assay [2] . We prepared a 10% acrylamide gel, and 15 μg/ml protein of each sample was loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
- Enzyme Assay and Kinetics
Purified enzyme was reacted with L -asparagine using Nessler’s reagent to measure the amount of ammonia released [7] . Ammonium sulfate solution was diluted to 1.2, 2.4, 3.6, 4.8, and 6 μmol/ml to plot the standard curve. The reaction contained 50 mM Tris-Cl (pH 8.6) with 0.25-10 mM L -asparagine. The reaction was terminated after 30 min by adding 50 μl of ice-cold 1.5 M trichloroacetic acid. The solution was centrifuged at 13,200 rpm for 2 min to precipitate the pellet. A final volume of 200 μl was reacted with 500 μl of Nessler’s reagent in 4.3 ml of distilled water. The resulting color change of the reaction was measured at an optical density of 436 nm. All sets of L -asparaginase activity assays were triplicates. All experiments were performed in triplicates. The t -test was performed to validate the statistical significance of different groups using R software.
- Determination of the Oligomeric State of Purified SoAsn
To determine the oligomeric state of purified SoAsn, a standard curve was made with the known size of the proteins alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), and lysozyme (14 kDa), using a Sephacryl S-200 16/60 (GE Healthcare Life Science). These standard molecules exists in monomeric states and thus display individual molecular size.
- Effects of Various Parameters onL-Asparaginase Activity
We determined the effects of pH, temperature, metal ions, NaCl, and substrates on the activity of SoAsn and the results were compared with EcAsn. The reaction condition was identical to that described above. The activity was measured at 10 and 30 min. Metal solution stocks (100 mM) were prepared in Tris buffer (pH 8.6). The final concentrations of the metals were 2, 5, and 10 mM. To test the influence of salt stress, we dissolved NaCl in Tris buffer (pH 8.6) and diluted in the reaction mixtures (0.2, 0.5, 1, 1.5, and 2 M). To compare the substrate specificity, we reacted purified SoAsn and EcAsn with 10 mM of L -glutamine and L -asparagine.
- Phylogenetic Analysis and Sequence Comparison
To compare the amino acid sequence of SoAsn with homologous sequences, we obtained genetic information from the National Center for Biotechnology Information GenBank database. We analyzed the protein sequences using the Basic Local Alignment Search Tool (BLASTP). The multiple sequences were aligned using the CLUSTAL-W a lgorithm in MEGA 5.1. The phylogeny was inferred using the neighbor-joining method [6 , 24] . A bootstrap consensus tree with 500 replicated evolutionary distances was constructed using the p-distance method and showed the number of amino acid differences per site [23 , 30] . We included 27 species harboring L -asparaginase to correlate the phylogenetic association at the species level.
Results and Discussion
- Sequence Analysis to Determine Conserved Domain and Active Sites
SoAsn showed a maximum 33% and 32% amino acid sequence identity with the asparaginase from E. coli mutant T89v and E. chrysanthemi . Conserved domain sequences of the active binding sites are indicated in Fig. 1 A. The active sites were Gly10, Thr11, Ala57, Ser58, Glu59, Gly89, Thr90, Asp91, Lys165, and Ser260 that were located to the N-terminus over six separate regions, totaling 10 amino acids. Threonine at the active site is known to play a major role as a nucleophile. Water molecules can attack it and cleave the ammonia group. These active sites are preferably oriented toward the substrate in the enzyme-substrate complex. The catalytic binding sites are shown in 3D structure in Fig. 1 B.
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Conserved domain amino acids and active sites of L-asparaginase. (A) Comparison of conserved amino acid sequences among the L-asparaginase superfamily. The sequences were queried using the National Center for Biotechnology Information database. (B) Active sites of L-asparaginase. The dimeric structure is shown with the catalytic sites shaded in yellow.
- Phylogenetic Analysis ofL-Asparaginases from Diverse Species
The phylogenetic relationship of L -asparaginase from diverse species was analyzed. The criteria for selecting the genes were as follows: (i) Commercially used microbial sources such as E. coli , Erwinia , and Serratia , which are recognized as clinically approved therapeutic sources. (ii) The top 10 species in the BLASTP results. (iii) Species originating from a saline environment. Among 27 species in the phylogenetic tree, SoAsn was closely related to L -asparaginase from Staphylococcus aureus , followed by L -asparaginase from Bacillus licheniformis . The correlation might be attributed to common characteristics of gram-positive species, because L -asparaginases from gram-negative species are less closely related to gram-positive-originated L -asparaginases. The enzymes from pathogenic Salmonella and E. coli O157:H7 were particularly close. Although EcAsn was the most homologous gene in the BLASTP results, it did not have a close phylogenetic relationship with SoAsn. Therefore, the phylogeny of L -asparaginase was congruent with the species phylogeny.
- Enzyme Purification and Oligomeric State
SoAsn was induced with IPTG and expressed in E. coli BL21 (DE3) as a soluble enzyme. The results of the SDS-polyacrylamide gel electrophoresis analysis were consistent with the expected size of 37.5 kDa amino acid sequence, as shown in Fig. 2 A. The stepwise asparaginase activity is shown in Table 1 . Notably, E. coli BL21 cell extract activity reached a 97.75-fold increase on the Ni-NTA column, and a second purification using an Amicon filter combined with buffer and concentration changes reached a 128.47-fold increase. To determine the oligomeric state of purified SoAsn, we performed size-exclusion chromatography. The peak in the image in Fig. 2 B represents a dramatic increase in elution volume near 100~110 ml and demonstrates that native SoAsn was in a dimeric state. Type II L -asparaginase is typically a dimer or tetramer [1 , 20 , 30] . Of particular note, an extracellular asparaginase from Rhodosporidium toruloides has been reported to be a homodimer [3 , 13] .
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and the dimeric state of SoAsn. (A) SDS-PAGE analysis of cell extracts and purified L-asparaginase. Lane P, protein marker; lane 1, control lysate; lane 2, control solution; lane 3, 0.5 mM isopropyL-β-D-thiogalactopyranoside (IPTG)-induced lysate; lane 4, IPTG-induced soluble product; lane 5, purified SoAsn enzyme. (B) Determination of the oligomeric state of purified SoAsn.
Purification steps and stepwise calculation ofL-asparaginase activity.
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Escherichia coli BL21 (DE3) cells were treated with isopropyl-β-D-thiogalactopyranoside to induce L-asparaginase expression. After purification with affinity chromatography on a nickel-nitrilotriacetic acid (Ni-NTA) column, the sample was concentrated with an Amicon filter and buffer change. The enzyme assay was properly diluted at each step in a 50 μl volume, and enzyme activity was measured. The measured activity was converted to specific activity units (U/mg) to calculate the purification fold change.
- Enzyme Kinetics ofL-Asparaginase
The kinetic properties of SoAsn were compared with other asparaginases ( Table 2 ). Previous studies of biochemical properties and structure have shown that the K m value of EcAsn was smaller than that of L -asparaginase from other microbial sources [7 , 9 , 17] . A low K m value indicates high substrate affinity, which is desirable for enzyme efficiency; however, V max was difficult to compare among species owing to the unequal amount of substrate in the previous studies. We conducted a substrate-dependent L -asparaginase activity assay of purified SoAsn, measuring the K m v alue according to the Michaelis-Menten equation. The K m value of SoAsn was 2.2 mM, which was similar to that of Tetrahymena pyriformis . The membrane-bound L -asparaginase of T. pyriformis had a sensitivity similar to that of SoAsn. From this measurement, we determined the maximum velocity (V max ) and catalytic efficiency constant (K cat ). V max of SoAsn was 61.4 U/ml and K cat was 2.7 × 10 2 / min (4.65/s). The maximum velocity, which is dependent on enzyme concentration, was retained when the substrate concentration increased to more than 4.5 mM.
Microbial sources ofL-asparaginase and properties.
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Basic biochemical properties of major bacteria sources of L-asparaginase were compared with respect to temperature optima, pH optima, Km, specific activity, and molecular mass.
- L-Asparaginase Activity in Various pH
The pH profile of L -asparaginase is illustrated in Fig. 3 . The maximal activities of SoAsn and EcAsn were observed at pH 9~10. SoAsn retained 80% of its maximal activity in the range of pH 8~9; however, its activity was dramatically decreased to 20% below pH 8, whereas EcAsn retained 67.5% activity at neutral pH and maintained 30% activity at pH 4. SoAsn was more sensitive than EcAsn under acidic conditions. The comparison between SoAsn and EcAsn demonstrated that although the enzymes had optimal activity within the same pH range, the pH range of SoAsn was more narrower.
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Effect of pH on L-asparaginase from SoAsn and EcAsn. Closed squares represent SoAsn and open squares represent EcAsn. Asterisks indicate the statistical difference determined by t-test (p < 0.05).
- Effect of Temperature onL-Asparaginase
We measured the influence of temperature on enzyme activity between 4℃ and 70℃ ( Fig. 4 ). The results indicated that SoAsn and EcAsn exhibited maximum enzyme activity between 25℃ and 37℃. Below 25℃, SoAsn lost most of its activity, whereas EcAsn retained 60% of its activity. Above 50℃, SoAsn and EcAsn exhibited similar activity changes. SoAsn had optimal thermal stability between 37℃ and 50℃, which was comparable to that of EcAsn. The thermal stability of SoAsn between 50℃ and 70℃ was similar to that of EcAsn. However, the enzyme stability range of SoAsn was more defined than that of EcAsn within the range of 4℃-37℃. Therefore, at extreme temperatures, EcAsn was more stable than SoAsn even though their optimal temperatures were similar.
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Effect of temperature on L-asparaginase activity. (A) Thermal stability over 30 min at each temperature. (B) Enzyme activity during a 10 min reaction at each temperature. Asterisks indicate the statistical difference determined by t-test (p < 0.05).
- Effects of Metal Cofactors onL-Asparaginase Activity
To enhance L -asparaginase activity with cofactor binding at the catalytic site of the enzyme, we conducted experiments using metals as cofactors. Other studies of metal dependency have used concentrations of 2 mM [29 , 34] , so we explored higher metal ion concentrations from 2 to 10 mM. Among eight metal ions tested (K + , Ca 2+ , Ni 2+ , Cu 2+ , Co 2+ , Mg 2+ , Zn 2+ , and Fe 2+ ), cobalt was the strongest cofactor ( Fig. 5 ). Cobalt enhanced SoAsn activity to 263.9%, iron increased the activity to 203.7%, and a slight increase was observed with copper (109.3%). Magnesium showed distinct metal-dependent activity, strongly inhibiting SoAsn (25%) but greatly increasing EcAsn activity (330.6%). Calcium treatment resulted in a complete inhibitory effect on SoAsn activity. Repeating the test with increasing concentrations of metal ions showed that SoAsn was distinct in its requirements for metal cofactors. L -Asparaginase from Rhizomucor miehei was enhanced by Ca 2+ and Mn 2+ (to 144% and 183%, respectively) [32] . On the contrary, Co 2+ , Fe 3+ , and Cu 2+ reduced the activity of L -asparaginase from R. miehei to 69%, 41%, and 13%, respectively [32] . We verified that 2 mM ethylenediaminetetraacetic acid inhibited activity to 16%, suggesting that SoAsn was dependent on the metals.
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Effects of metal ions on L-asparaginase activity. K+, Ca2+, Ni2+, Cu2+, Co2+, Mg2+, Fe2+, and Zn2+. Asterisks indicate the statistical difference determined by t-test (p < 0.05). ND: Not detected.
- Effect of Salt Stress onL-Asparaginase Activity
We hypothesized that SoAsn from halotolerant Staphylococcus sp. OJ82 would have considerable activity under salt stress conditions. The structures of the enzyme could change in the presence of salt stress via surface charge interaction. We speculated that under optimal conditions, SoAsn would display resistance to saline conditions. We hypothesized that cofactor binding at catalytic sites and optimized pH would stabilize the hydrogen bonds and the nucleophilic interaction of the substrate-enzyme complex against salt stress. Thus, under optimized condition, the enzyme was expected to be more resistant to NaCl inhibition because the substrate binds more actively at the catalytic sites. With respect to growth conditions, 2% (w/v) of NaCl in the media maximizes enzyme production by E. coli [9] . Mucor -originated L -asparaginase activity was unaffected by the addition of 4% (w/v) NaCl [8 , 23] . The enzyme-substrate complex was examined under salt stress up to 2 M NaCl, which was comparable to 11.5% (w/v). At all concentrations, SoAsn exhibited enzyme activity greater than that of EcAsn ( Fig. 6 ). SoAsn retained 61.2% of its initial activity at the NaCl concentration of 2 M, whereas EcAsn activity was reduced to 48%. These results demonstrated that compared with EcAsn, SoAsn retained greater salt tolerance. The optimized condition for maximal activity was 2 mM cobalt in a solution with pH 9 at 37℃ for 30 min. We verified that optimized SoAsn exhibited a 4.2-fold increase in activity compared with that under the standard condition. In contrast to our assumption that optimized conditions would confer salt resistance to the enzyme, SoAsn activity decreased to less than 50% under these conditions. Optimized conditions did not provide salt tolerance to the enzyme, and this result was attributed to the structure of SoAsn under optimal conditions and the interaction between high NaCl concentration and enzyme binding sites. The conformational changes induced by cofactor binding and optimized pH seem to be affected under salt stress. Halophilic enzymes were tightly bound by sodium rather than by substrate [20] . We speculated that salt stress would induce loop flexibility in the enzyme structure that would be deeply involved in enzymatic activity [22 , 33] . The mobile structure of L -asparaginase gives it flexible access to the substrate, thus increasing enzyme activity. However, a salt bridge between NaCl and cobalt-bound L -asparaginase makes the enzyme rigid, preventing the binding of the enzyme to substrate [21 , 24] . Halophilic enzymes have a high negative surface charge and are thus dissociated easily and become more flexible in the presence of NaCl. On the contrary, non-halophilic enzymes tend to aggregate under high-salt conditions [19] . Furthermore, the affinity of NaCl to catalytic sites of halophilic enzymes is lower, which results in stronger binding to salts than to non-salts [4] . As for external stress, the hydrophobic surface accounts for the resistance to detergents (SDS). Compared with enzyme from E. coli and E. carotovora , enzyme produced by S. marcescens was more resistant to dissociation by SDS, perhaps because the Serratia enzyme has relatively high hydrophobicity [13] .
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Effect of salt stress (NaCl) on L-asparaginase activity. The asterisk indicates the statistical difference determined by t-test (p < 0.05).
- Substrate Specificity
We verified that L -asparaginase has specificity for the amide-containing substrate L -asparagine against L -glutamine. SoAsn and EcAsn were reacted with L -asparagine and L -glutamine, and the relative L-glutaminase activity was compared against L -asparaginase activity, which was designated as 100%. SoAsn had higher glutaminase activity (58.2%) than EcAsn (19%). This result indicated that the substrate specificity of SoAsn was lower than that of EcAsn. The substrate specificity of L -asparaginase is crucial in antileukemia therapy. The unnecessary degradation of glutamine is the cause of immunogenicity [32] . To date, the most promising approach to extending half-life in the plasma and reducing the immunogenicity of therapeutic proteins is to couple them covalently to PEG [13 , 34] . Surface protein residues are covalently linked with PEG, masking surface sites and enlarging molecular size and thereby enhancing steric hindrance. PEGylation is a strategy for increasing substrate specificity [24 , 33] . We conducted PEG-asparaginase experiments with 1%, 2.5%, and 5% (v/v) PEG 4000. The results indicated that all PEG-treated samples had increased L -asparaginase activity (data not shown). Substrate specificity was noticeably improved with 2.5% PEG treatment: the glutaminase activity of SoAsn was reduced to 31% from 59%. Compared with non-PEG-treated samples, which exerted more than 50% glutaminase activity, 2.5% PEG-treated samples gained substrate specificity.
Although numerous studies have isolated and investigated asparaginases, little has been explored about salt-stress-related L -asparaginase activity. This is a particularly significant report of molecular cloning of L -asparaginase from gram-positive Staphylococcus sp. OJ82, because most L -asparaginase originates from gram-negative bacteria or fungi. Specifically, the purified SoAsn retained 61.2% activity in 2 M NaCl, which is 48% higher than the activity of commercial EcAsn. We demonstrated that SoAsn can withstand salt stress, as Staphylococcus sp. OJ82 was viable under high-salt conditions. Use of cobalt as a metal cofactor for SoAsn was the major distinction, whereas other L -asparaginases were enhanced by magnesium or zinc. Although SoAsn and EcAsn are distantly related in the phylogenetic tree, the optimal temperature and pH conditions for their activity are similar. SoAsn was more sensitive under extremes of pH and temperature, but EcAsn was relatively stable under acidic conditions and at room temperature. To use SoAsn efficiently without side effects, we tried to improve its substrate specificity. In further studies, we will engineer the protein by replacing catalytic residues and alternating the surface charge to increase the substrate specificity. We recommend a study for safety validation to potentiate this enzyme as an effective drug for the treatment of leukemia and an anti-acrylamide additive in the food industry.
This work was supported by a grant from the Next-Generation BioGreen 21 Program (PJ0082082013) of the Rural Development Administration, Republic of Korea.
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