Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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A Newly Identified Glutaminase-Free L-Asparaginase (L-ASPG86) from the Marine Bacterium Mesoflavibacter zeaxanthinifaciens

  • Lee, Su-Jin (Jeju International Marine Science Research & Education Center, Korea Institute of Ocean Science & Technology) ;
  • Lee, Youngdeuk (Jeju International Marine Science Research & Education Center, Korea Institute of Ocean Science & Technology) ;
  • Park, Gun-Hoo (Jeju International Marine Science Research & Education Center, Korea Institute of Ocean Science & Technology) ;
  • Umasuthan, Navaneethaiyer (Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University) ;
  • Heo, Soo-Jin (Jeju International Marine Science Research & Education Center, Korea Institute of Ocean Science & Technology) ;
  • Zoysa, Mahanama De (College of Veterinary Medicine, Chungnam National University) ;
  • Jung, Won-Kyo (Department of Biomedical Engineering and Centre for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National University) ;
  • Lee, Dae-Won (Marine Ecosystem and Biological Research Center, Korea Institute of Ocean Science & Technology) ;
  • Kim, Hanjun (Marine Ecosystem and Biological Research Center, Korea Institute of Ocean Science & Technology) ;
  • Kang, Do-Hyung (Jeju International Marine Science Research & Education Center, Korea Institute of Ocean Science & Technology) ;
  • Oh, Chulhong (Jeju International Marine Science Research & Education Center, Korea Institute of Ocean Science & Technology)
  • Received : 2015.10.28
  • Accepted : 2016.03.08
  • Published : 2016.06.28
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Abstract

L-Asparaginase (E.C. 3.5.1.1) is an enzyme involved in asparagine hydrolysis and has the potential to effect leukemic cells and various other cancer cells. We identified the L-asparaginase gene (L-ASPG86) in the genus Mesoflavibacter, which consists of a 1,035 bp open reading frame encoding 344 amino acids. Following phylogenetic analysis, the deduced amino acid sequence of L-ASPG86 (L-ASPG86) was grouped as a type I asparaginase with respective homologs in Escherichia coli and Yersinia pseudotuberculosis. The L-ASPG86 gene was cloned into the pET-16b vector to express the respective protein in E. coli BL21 (DE3) cells. Recombinant L-asparaginase (r-L-ASPG86) showed optimum conditions at 37-40℃, pH 9. Moreover, r-L-ASPG86 did not exhibit glutaminase activity. In the metal ions test, its enzymatic activity was highly improved upon addition of 5 mM manganese (3.97-fold) and magnesium (3.35-fold) compared with the untreated control. The specific activity of r-L-ASPG86 was 687.1 units/mg under optimum conditions (37℃, pH 9, and 5 mM MnSO4).

Keywords

Introduction

L-Asparaginase is a member of the homologous amidohydrolase family that catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia [7]. This enzyme, which is distributed in diverse sources such as animals [28], plants [42], algae [37], fungi [40], and bacteria [38], exists in two types: I and II. Type I asparaginase is a low-affinity enzyme found in the cytoplasm, whereas type II asparaginase is a high-affinity enzyme [36] identified within the periplasmic space [9].

L-Asparaginase was the first enzyme discovered with antileukemic activity [41]. The antitumor activity of asparaginase was first observed in guinea pig serum [21] and elucidated the asparagine depletion by asparaginase [7]. Asparagine is an amino acid that is required for protein synthesis and survival. Most normal cells have asparagine synthetase for the synthesis of asparagine from aspartate and glutamine. However, some tumor cells are dependent on an exogenous source of asparagine for protein synthesis, because tumor cells lack or have very low levels of asparagine synthetase [8]. Therefore, depletion of asparagine by L-asparaginase leads to the selective death of tumor cells. Currently, L-asparaginase from Escherichia coli and Erwinia chrysanthemi is being used for the clinical treatment of acute lymphoblastic leukemia (ALL) and non-Hodgkin’s lymphoma [25,31]. Many countries use asparaginase from one of these sources as an antileukemic drug [1]. However, the glutaminase activity of L-asparaginase causes side effects, such as liver dysfunction, pancreatitis, leucopenia, neurological seizures, and coagulation abnormalities [11]. Asparaginase is also used in the food industry during food processing to reduce acrylamide [22]. Acrylamide, which is formed from asparagine when starchy foods such as breads and cookies are baked or fried, is known to have a toxic effect on the nervous system and fertility [44]. Acrylamide, a potential carcinogen, results from the reaction between asparagine and reducing sugars found in carbohydrate-rich foods when they are processed above 180℃ [32]. Pretreatment with L-asparaginase prevents acrylamide formation in carbohydrate-rich foods during the frying or baking process [16,35].

In this study, we identified the L-asparaginase (L-ASPG86) gene from Mesoflavibacter zeaxanthinifaciens and produced the recombinant protein to characterize its enzymatic properties.

 

Materials and Methods

Analysis of L-ASPG86 Gene

M. zeaxanthinifaciens strain S86 was isolated from the seawater of Chuuk State in Micronesia and the genome sequenced in our previous study [34]. The L-ASPG86 gene was identified from the genome of M. zeaxanthinifaciens, and its homology and domain structure were examined using the protein BLAST of the NCBI (http://blast.ncbi.nlm.nih.gov). The molecular mass and isoelectronic point (pI) were predicted by the Compute pI/Mw tool (http://web.expasy.org/compute_pi/). Multiple sequence alignment of the L-asparaginase sequences was performed using ClustalW [45]. A phylogenetic tree was constructed for L-asparaginase amino acid sequences using the neighbor-joining (NJ) method from MEGA 6.0 [43]. The bootstrap values were replicated 1,000 times to obtain the confidence values for a robust analysis. Homology search in the PDB database using BLAST and PSIBLAST was performed to find a template structure. The 3D model of the L-ASPG86 was predicted using the Swiss Model Server [4], using as template E. coli (PDB code 2P2N). The 3D structural models were visualized using PyMOL [10].

Cloning of L-ASPG86 Coding Sequence

M. zeaxanthinifaciens S86 was cultivated after growing in marine broth (BD Biosciences, USA) at 20℃ for 2 days. Genomic DNA was extracted with the AccuPrep Genomic DNA Extraction Kit (Bioneer, Korea). PCR was performed to amplify the full-length ORF of L-ASPG86 from genomic DNA of M. zeaxanthinifaciens S86 using a pair of primers (forward primer 5’-gagacatATGGGTAAAAGAGCTAGAATATTA-3’ with NdeI site, and reverse primer 5’-gagactcgagttaATTTTGGGACATCTCCCC-3’ with XhoI site and stop codon). The PCR conditions were as follows: initial denaturation at 94℃ for 5 min; denaturation at 94℃ for 30 sec, annealing at 48℃ for 30 sec, and extension at 72℃ for 60 sec for 30 cycles; and a final extension at 72℃ for 5 min. The product was purified by the AccuPrep Gel Purification Kit. The purified PCR product and pET-16b vector (Novagen, USA) were digested with NdeI (Takara, Japan) and XhoI (Takara) restriction enzymes, and ligated with T4 DNA ligase (Takara). The ligation product was transformed into E. coli DH5α cells and purified with the AccuPrep Nano-Plus Plasmid Mini Extraction Kit (Bioneer). The recombinant plasmids were confirmed by sequencing.

Overexpression and Purification of r-L-ASPG86

The plasmid was transformed into E. coli BL21 (DE3) and a positive colony was inoculated onto 4 ml of LB broth containing ampicillin (100 μg/ml) and incubated overnight at 37℃ in a shaking incubator. The overnight culture was transferred to 250 ml of LB broth containing ampicillin and incubated until the optical density at 600 nm reached 0.6. The r-L-ASPG86 expression was induced by addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 24 h at 30℃ with shaking at 200 rpm. Thereafter, cells were collected by centrifugation at 8,000 rpm for 10 min at 4℃. The cell pellets were resuspended in binding buffer and sonicated by a Q700 sonicator (Qsonica, USA). The lysate was centrifuged at 13,000 rpm for 30 min at 4℃, and the supernatant was collected. The r-L-ASPG86 was purified using a His-Bind kit (Novagen, USA) according to the manufacturer’s instruction. The purified r-L-ASPG86 was keep at -20℃ with 20% glycerol until use. Purified r-L-ASPG86 was examined by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with standard marker proteins of known molecular weight. Protein concentration was determined using a BCA protein assay kit (Pierce, USA).

L-Asparaginase Activity Assay for r-L-ASPG86

The L-asparaginase enzyme assay was performed by using Nessler’s reaction [17]. Briefly, 1 ml of the total reaction mixture consisted of 1 μg of purified enzyme solution, 50 mM Tris-HCl (pH 8.6), and 10 mM L-asparagine and was incubated at 37℃ for 10 min. The reaction was stopped by adding 50 μl of 1.5 M trichloroacetic acid. After centrifugation at 10,000 rpm for 5 min, 0.2 ml of the supernatant was mixed with 3.8 ml of distilled water and 0.5 ml of Nessler`s reagent. The absorbance was measured at 450 nm using a spectrophotometer. A standard curve of ammonium sulfate was used for calculating the ammonia concentrations. One unit of the L-asparaginase activity was defined as the amount of enzyme that liberates 1 μmol of ammonia per minute. Glutaminase activity was assayed by the same method using L-glutamine as substrate.

Biochemical Characterization of Purified r-L-ASPG86

The optimum temperature for the enzyme activity was determined by incubating the assay mixture for 10 min at temperatures ranging from 30℃ to 80℃, including 37℃, in Tris-HCl buffer (pH 8.6). Effects of pH on enzyme activity were evaluated by measuring the activity for 10 min at 37℃ with different buffers as follows; citrate-phosphate (pH 3.0-7.0), sodium-phosphate (pH 6.0-8.0), Tris-HCl (pH 8.0-9.0), and glycine-NaOH (9.0-10). To determine the effect of metal ions and chelating agent on the activity of r-L-ASPG86, the enzymatic activity was assayed in the presence of various metal ions and chelating agents (CaCl2, CuSO4, FeSO4, KCl, MgSO4, MnSO4, NaCl, ZnSO4, and EDTA) with 1 or 5 mM final concentrations. The thermostability of r-L-ASPG86 was investigated by pre-incubating at 40℃ or 50℃ for 1 h (in 10 min intervals). The specific activity was determined at optimal conditions.

 

Results

Sequence Characterization of L-ASPG86

An asparaginase homolog was identified from M. zeaxanthinifaciens S86 and named L-ASPG86 (GenBank Accession No. KP705084). The nucleotide and amino acid sequences are shown in Fig. 1. The L-ASPG86 gene contains a 1,035 bp open reading frame (ORF) and encodes a putative protein of 344 amino acid (aa) residues. The predicted molecular mass and isoelectric point (pI) are 38 kDa and 6.6, respectively. A conserved asparaginase type I domain was found between aa 6 and 342. Active site residues (aa 14-15, 60-61, 90-92, 117, 168 aa) and homodimer interface sites (15, 25-27, 56, 59-65, 67-68, 92-93, 96, 118-119, 168-173, 217-227, 229-230, 233, 236-237, 247, 249-251, 255, 279, 282-286, 290, 309, 313, and 317 aa) were also predicted. To determine the relationship between L-ASPG86 and other known asparaginase members from various bacteria, a phylogenetic tree was constructed (Fig. 2). The phylogenetic tree consisted of two main clades: glutaminases (E.C. 3.2.1.2) and asparaginases (E.C. 3.2.1.1) with glutaminase-asparaginase (E.C. 3.2.1.38). The asparaginasee clade was further divided into two subclades, namely asparaginases and glutaminase-asparaginases, the first of which was again divided into two groups: type I and type II asparaginases. The L-ASPG86 grouped within the type I asparaginase clade with respective homologs in E. coli and Y. pseudotuberculosis. L-ASPG86 was most closely related, with 41.4% identity, to E. coli type I L-asparaginase. The three-dimensional (3D) modeling results indicate that L-ASPG86 exists as a homotetramer. Each of the five active sites is located at the N-terminal domains (Fig. 3).

Fig. 1.Nucleotide and deduced amino acid sequences of L-asparaginase from M. zeaxanthinifaciens S86. Large box, L-asparaginase type I domain; dot boxes, active site; underlines, residues at the homodimer interface.

Fig. 2.Phylogenetic analysis of amino acid sequences of L-ASPG86 with other known characterized asparaginase and glutaminase counterparts. Phylogenetic analysis was conducted by the NJ method using MEGA 6.0, based on sequence alignment using ClustalW. The numbers indicate the bootstrap confidence values of 1,000 replicates. The accession numbers of L-asparaginase homologs are given in parentheses. Bar, 0.2 substitutions per amino acid position.

Fig. 3.Three-dimensional models of L-ASPG86. (A) L-ASPG86 tetramer with identical subunits colored cyan, yellow, green, and pink. (B, C) A representation of the L-ASPG86 monomer. α-Helices are shown in red and β-strands in yellow. The active site residues are shown in blue.

Expression and Purification of r-L-ASPG86

IPTG-induced overexpression of the His-tagged L-ASPG86 fusion protein was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Fig. 4). A strong single band similar in mass to the predicted molecular mass of r-L-ASPG86 (41 kDa, including the 6-His tag) was detected, corresponding to the purified recombinant protein. This confirmed successful overexpression of r-L-ASPG86 along with sufficient purity and integrity of the eluted fusion protein.

Fig. 4.SDS-PAGE analysis of r-L-ASPG86. Protein samples were separated on 12% SDS-PAGE and stained with Coomassie brilliant blue. M, molecular mass marker (Takara, Japan); lane 1, total cellular extract from E. coli BL21 (DE3) before IPTG-induction; lane 2, total cellular extract after induction; lane 3, purified r-L-ASPG86.

Biochemical Characterization of Purified r-L-ASPG86

We investigated the relative activity of r-L-ASPG86 over a range of temperatures (between 30℃ and 80℃ with 10℃ intervals, including 37℃). r-L-ASPG86 showed the highest enzymatic activity at 40℃, and similar activity was detected at 37℃ (Fig. 5A). Its activity decreased gradually from 50℃. In the optimum pH assay, the highest activity was observed at pH 9, and more than 60% of the relative activity was retained within the pH range of 7-10, as shown in Fig. 5B. The effects of metal ions and EDTA on r-L-ASPG86 activity are shown in Fig. 5C at two different concentrations (1 and 5 mM). r-L-ASPG86 was strongly inhibited by both the zinc ion and EDTA. However, r-L-ASPG86 demonstrated increased activity upon addition of 5 mM manganese (3.97-fold) and 5 mM magnesium (3.35-fold) ions compared with the untreated control. The thermal stability of r-L-ASPG86 was examined following pre-incubation with the enzyme at 40℃ or 50℃ for 60 min (Fig. 5D). At 40℃, the activity of r-L-ASPG86 was reduced by approximately 50% after 20 min and then decreased over time. However, r-L-ASPG86 was completely inactivated after a 10-min incubation at 50℃. Specific activity was tested under optimal conditions (40℃, pH 9, and 5 mM manganese); r-L-ASPG86 had a specific activity of 687.1 units/mg. Glutaminase activity of r-L-ASPG86 was not detected under the tested conditions (data not shown).

Fig. 5.Biochemical properties of purified r-L-ASPG86. (A) Effects of temperature on r-L-ASPG86 activity at temperature in the range of 30-80℃. (B) Effects of pH on r-L-ASPG86 activity at pH in the range of 3-10. (C) Effects of metal ions and chelating agents on r-L-ASPG86 activity at 1 and 5 mM, measured after incubation at 37℃ for 10 min. (D) Effects of thermostability on remaining r-L-ASPG86 activity at 40℃ and 50℃ for 0-60 min.

 

Discussion

In this study, we identified an asparaginase gene (L-ASPG86) from the M. zeaxanthinifaciens S86 genome. The identified gene was cloned, expressed, and characterized. This is the first enzymatic characterization of an asparaginase isolated from the family Flavobacteriaceae. In our study, we found that the amino acid sequence of L-ASPG86 contains a highly conserved L-asparaginase type I domain with a homodimer interface. Phylogenetic analysis grouped L-ASPG86 into a type I asparaginase with E. coli and Y. pseudotuberculosis (Fig. 2). These results suggest that L-ASPG86 belongs to a type I asparaginase and is localized in the cytoplasm. Furthermore, the presence of a homodimer interface suggests that L-ASPG86 may be found as a homodimer upon a protein BLAST search on the NCBI website (Fig. 1). However, the 3D structure of L-ASPG86 (Fig. 3) was predicted as a homotetramer that was most closely related to E. coli type I L-asparaginase, with 41.4% similarity. The structure of the E. coli type I L-asparaginase analyzed by X-ray crystallography suggested a dimer of two intimate dimers [48]. It is also possible that L-ASPG86 is a homotetramer as an E. coli type I L-asparaginase.

Most of the reported bacterial asparaginases, such as those from Erwinia carotovora [19], Staphylococcus sp. [15], Pectobacterium carotovorum [26], Penicillium brevicompactum [13], Pseudomonas aeruginosa [12], and Pseudomonas stutzeri [30], have exhibited optimal activity between 30℃ and 40℃. The r-L-ASPG86 produced here also demonstrated its highest activity at 37-40℃, which is similar to those of other bacteria (Table 1). r-L-ASPG86 showed optimal activity at pH 9, which is similar to homologs from Rhizobium etli [3], Staphylococcus sp. [15], Pseudomonas aeruginosa [12], Pseudomonas stutzeri [30], and Pyrococcus furiosus [5] (Table 1). It also retained more than 60% of its activity at a pH range between 7 and 10 (Fig. 5B). Upon addition of metal ions and the chelate effect, r-L-ASPG86 activity was highly enhanced in the presence of Mn2+ and Mg2+ (Fig. 5C).

Table 1.aRelative glutaminase activity: compared against L-asparaginase activity. bNT: not tested. cND: not detected.

The activity of bacterial asparaginases is dramatically increased in the presence of cations. For instance, Na+ and K+ increase the asparaginase activity in Bacillus coagulans [27], Pseudomonas stutzeri [30], and Erwinia carotovora [46]. Meanwhile, Mg2+ enhances the asparaginase activity in bacterial species, such as Acinetobacter calcoaceticus [18] and Pseudomonas stutzeri MB-405 [30]. Similarly, the activity of r-L-ASPG86 was strongly enhanced by Mg2+; however, its activity was inhibited by 5 mM Na+. r-L-ASPG86 was not significantly affected by the K+ ion. Interestingly, r-L-ASPG86 showed the highest asparaginase activity in the presence of 5 mM Mn2+. In previous studies, only the activity of an asparaginase from Streptomyces sp. [6] was reported to be enhanced by Mn2+. In our analogous study to clone and characterize the endo-β-1,3-glucanase from M. zeaxanthinifaciens S86, we found that its activity was strongly enhanced by 5 mM Mn2+. This observation suggests that Mn2+ may be an important metal that affects enzymatic functions in M. zeaxanthinifaciens.

L-Asparaginase can be used for the treatment of ALL [14,29]. However, cancer treatment with asparaginases has been hampered by their potent glutaminase activity, which can cause liver dysfunction, pancreatitis, leucopenia, neurological seizures, and coagulation abnormalities, leading to intracranial thrombosis or hemorrhage [11]. Therefore, glutaminase-free asparaginase is necessary for medical treatment in humans. In this study, the assay for determining the substrate-specific activity of r-L-ASPG86 showed that it possesses only L-asparaginase activity and not glutaminase activity. Taken together, the biochemical properties of r-L-ASPG86 are similar to those of previously reported L-asparaginases that are used as therapeutic agents in the treatment of ALL. Generally, type I L-asparaginases display high Km values toward L-asparagine and show catalytic activity toward L-glutamine, whereas type II L-asparaginases exhibit affinity to L-asparagine and low-to-negative activity toward L-glutamine [49]. Interestingly, r-L-ASPG86 showed no activity toward L-glutamine, which is similar to previously reported type II asparaginases, although it showed the typical molecular features of a type I L-asparaginase.

In summary, L-ASPG86 contains type I asparaginase domain features with no signal peptide, which are typical molecular characteristics of type I L-asparaginases. Purified r-L-ASPG86 showed high catalytic activity against L-asparagine, but no glutaminase activity, which is comparable to other commercially available asparaginases used as anticancer agents. These results suggest that r-L-ASPG86 from M. zeaxanthinifaciens S86 could be a potential candidate enzyme to use in the treatment of ALL. Further studies are necessary to confirm its use as an anticancer therapeutic enzyme.

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