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Article

Characterization of L-Asparaginase Free of L-Glutaminase and Urease Activity Produced by the Marine Paraconiothyrium cyclothyrioides Strain MABIK FU00000820

by
Woon-Jong Yu
,
Ha Young Lee
,
Yong Min Kwon
,
Seung Seob Bae
,
Hyun-Ju Hwang
and
Dawoon Chung
*
Department of Biological Application and Technology, National Biodiversity Institute of Korea (MABIK), Seocheon 33662, Republic of Korea
*
Author to whom correspondence should be addressed.
Appl. Microbiol. 2025, 5(3), 100; https://doi.org/10.3390/applmicrobiol5030100
Submission received: 7 August 2025 / Revised: 13 September 2025 / Accepted: 15 September 2025 / Published: 17 September 2025
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Versions Notes

Abstract

Asparaginase (ASNase) is an important enzyme used to treat acute lymphoblastic leukemia. However, the clinical use of the currently available ASNases is limited because of their associated side effects. One of the major reasons for these adverse effects is the coactivity of glutaminase (GLNase) with ASNase. Furthermore, the concomitant urease activity may exacerbate the toxicity associated with ASNase treatment. Therefore, identifying novel sources of ASNase with minimal or no glutaminase and urease activities is important. We isolated a marine fungal strain, MABIK FU00000820, which produced ASNase free of GLNase and urease activity. Based on morphological and phylogenetic analyses, this strain was identified as Paraconiothyrium cyclothyrioides. The crude extract of intracellular ASNase exhibited the maximum activity at 37–50 °C, pH 8.5, and 0% (w/v) NaCl. In addition, the enzyme stability assay showed that the P. cyclothyrioides ASNase pre-treated at 4–25 °C for 2 h retained 77% of its activity compared to the untreated control. Based on the available literature, this appears to be the first study to investigate ASNase from P. cyclothyrioides, and it is of particular significance because the enzyme exhibits neither GLNase nor urease activity.

1. Introduction

L-Asparaginase (ASNase, EC number 3.5.1.1) is an enzyme used clinically for the treatment of acute lymphoblastic leukemia (ALL) and lymphosarcoma [1]. It catalyzes the conversion of L-asparagine to L-aspartic acid and ammonia. In cancer cells, ASNase depletes circulating asparagine, leading to impaired cell cycle progression, ultimately inducing cell death [2]. The use of ASNase in childhood ALL increases the survival rates by approximately 90% [3]. The therapeutic ASNase preparations currently in clinical use comprise Elspar®, Oncaspar®, Asparlas®, Erwinaze®, and Rylaze®, which are derived from the bacterial species Escherichia coli and Erwinia chrysanthemi [4]. However, these ASNases exhibit a wide range of side effects, including hypersensitivity and toxicities [5,6].
The concomitant L-glutaminase (GLNase) activity exhibited by ASNases is considered to be one of the main contributors to their adverse effects [5]. Molecular engineering approaches have been used to generate ASNases with reduced GLNase activity [7,8]. Strains of E. coli and E. chrysanthemi engineered via specific amino acid substitutions to produce ASNase variants with absent or reduced GLNase activity were investigated. However, because of the structural similarity between asparagine and glutamine, it is challenging to engineer ASNases with low to no GLNase activity while retaining antitumor efficacy [3]. In addition, secondary urease activity has been reported in commercial formulations of Escherichia coli ASNases [9]. Urease-mediated hydrolysis of urea in the bloodstream may result in ammonia toxicity. Accordinly, the exploration of alternative sources able to generate ASNase with negligible GLNase or urease activity is essential.
ASNases are produced by animals (e.g., mammals, birds, and fish), plants, and microorganisms, including bacteria and fungi. Microorganisms have gained considerable attention as sources of ASNase owing to their ease of large-scale production, purification, and manipulation [10]. Currently, E. coli and E. chrysanthemi are exclusively used to produce FDA-approved ASNases for therapeutic purposes. However, owing to the undesirable side effects of commercial ASNases, an increasing number of studies have focused on microbial ASNases that exhibit minimal or no GLNase and/or urease activity. For example, fungi, including Aspergillus niger, Chaetomium sp., Corprinopsis cinera, Meyerozyma guilliermondii, and Trichosporon asahii are known to produce ASNase free of GLNase and urease [11,12,13].
In this study, we characterized the intracellular ASNase activity of Paraconiothyrium cyclothyrioides strain MABIK FU00000820. This fungal strain was isolated from marine sediments and produces GLNase- and urease-free ASNase. Using the crude intracellular ASNase isolated from MABIK FU00000820, we optimized the temperature, pH, NaCl concentration, and incubation time for enzymatic activity. Moreover, the thermal stability of P. cyclothyrioides ASNase was investigated. Although the development of ASNase with reduced side effects is urgently needed, research on fungal ASNases has been relatively limited. In this context, the finding that a marine fungus produces ASNase lacking both GLNase and urease activities may provide fundamental insights for the development of novel ASNases.

2. Materials and Methods

2.1. Sample Collection, Fungal Isolation, and Cultivation

Marine sediment samples were collected from Ganghwa-gun, Incheon of the Republic of Korea (37°36′36.3″ N 126°31′13.8″ E), in January 2018 (during the winter season) and diluted with sterile distilled water. The diluted samples were plated onto potato dextrose agar (PDA; BD, USA) and yeast extract-peptone-dextrose (YPD; BD) agar, both supplemented with 0.01% (w/v) ampicillin (Sigma-Aldrich, St. Louis, MO, USA) and 0.01% (w/v) streptomycin (Sigma-Aldrich) and incubated at 25 °C for 7 d. Following incubation, fungal colonies were selected and subcultured on fresh PDA plates to obtain pure isolates. Fungal isolates were cryopreserved in 20% glycerol solution at −80 °C and maintained on PDA at 25 °C unless stated otherwise. The isolate MABIK FU00000820 was preserved in the Microbial Marine Bio Bank (MMBB) at the National Marine Biodiversity Institute of Korea (MABIK).

2.2. Assessment of ASNase, GLNase, and Urease Activities in Marine Fungi

Fungal isolates were screened for L-asparaginase (ASNase), glutaminase (GLNase), and urease activities using established methods [11,14]. The strains were cultured on modified Czapek-Dox (MCD) agar. The formulation (1 L) consisted of glucose (2.0 g), L-asparagine monohydrate (10.0 g; Sigma-Aldrich), KH2PO4, (1.52 g), KCl (0.52 g), MgSO4·7H2O (0.52 g), and trace elements, including 0.05% (w/v) each of CuNO3·3H2O, ZnSO4·7H2O, and FeSO4·7H2O, along with 15.0 g agar. The final pH was adjusted to 6.0 using 1 M NaOH. Phenol red (PR) was first prepared as a 2% (v/v) ethanolic stock solution and subsequently added to the culture medium to achieve a final concentration of 0.005% (v/v) as a pH indicator. For the detection of GLNase and urease activities, L-glutamine and filter-sterilized urea were used in place of L-asparagine, respectively, while NaNO3 was used in a separate control medium. All experiments were performed in triplicates.

2.3. Genomic DNA Preparation, Polymerase Chain Reaction, and Phylogenetic Studies

Genomic DNA (gDNA) was extracted by culturing strain MABIK FU00000820 in potato dextrose broth (PDB) at 25 °C for 7 d with shaking at 150 rpm. Mycelia were harvested and gDNA was isolated using the phenol:chloroform extraction method described by Chung et al. (2019) [15].
To identify strain MABIK FU00000820 at the molecular level, four genetic markers—the internal transcribed spacer (ITS) region, the partial D1/D2 domain of a large subunit (LSU, 28S) of rDNA, beta-tubulin(tub2), and a partial sequence of the translation elongation factor 1–α (tef1) gene—were amplified and analyzed. The ITS region was amplified using the primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) as described by [16]. The D1/D2 region of the large subunit (28S) rDNA was amplified using the primers LROR (5′-ACCCGCTGAACTTAAGC-3′) and LR5 (5′-TCCTGAGGGAAACTTCG-3′) [17]. For amplifying the tub2 regions, the primers Bt2a (5′-GGTAACCAAATCGGTGCTGCTTTC-3′) and Bt2b (5′-ACCCTCAGTGTAGTGACCCTTGGC-3′) were used [18]. The tef1 region was amplified using the primer pair EF1-983F (5′-GCYCCYGGHCAYCGTGAYTTYAT-3′) and EF1-2218R (5′-ATGACACCRACRGCRACRGTYTG-3′) following the method of [19]. PCR amplicons were cleaned with a purification kit (Qiagen, Hilden, Germany), and sequencing was performed in both directions by Macrogen Inc. (Seoul, Republic of Korea).
The amplified ITS, D1/D2 domain, tub2, and tef1 gene fragments were assembled using Geneious v9.0.5 software. The sequence similarity of MABIK FU00000820 was assessed by performing a BLASTN search (version 2.16.0) (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 7 January 2025) using the ITS, D1/D2 domain, tub2, and tef1 gene sequences as queries. Phylogenetic trees based on these genes were constructed in MEGA X [20] using the Kimura 2-parameter model and the neighbor-joining method. Clade support was evaluated using 1000 bootstrap replicates [21].

2.4. Preparation of Crude Enzyme Extract

To prepare the crude enzyme extract, 100 µL of MABIK FU00000820 fungal suspension (1.62 × 106 conidia/mL) was inoculated into 200 mL of PDB and incubated for 5 d at 25 °C with shaking at 150 rpm. After incubation, mycelia were collected by filtration through a single layer of autoclaved Miracloth (Merck Millipore, Darmstadt, Germany). The harvested mycelia were washed with MCD broth that did not contain agar or a pH indicator and then transferred into 200 mL of MCD broth to induce ASNase production. To determine the optimal cultivation time for maximizing ASNase activity, the mycelia were incubated in submerged fermentation at 25 °C and 150 rpm for 18, 24, 48, and 72 h.
For the further characterization of ASNase, the washed mycelia were incubated for 2 d (optimal cultivation time) at 25 °C with shaking at 150 rpm. After incubation, mycelia were collected by filtration through a single layer of autoclaved miracloth. The harvested mycelia were rinsed twice with 50 mM Tris-HCl buffer (pH 8.0) and subsequently resuspended in 30 mL of the same buffer. The fungal suspension was kept on ice during sonication, which was performed for 20 min in 30 s cycles. After sonication, the lysate was centrifuged at 11,000 rpm for 15 min, and the resulting supernatant was used as the crude enzyme extract to assess the ASNase activity of MABIK FU00000820 [22].

2.5. Determination of ASNase Activity Levels

ASNase activity was determined by measuring the ammonia released during the enzymatic reaction using the Nesslerization method [14,23,24]. The reaction mixture consisted of 100 µL of 0.04 M L-asparagine prepared in 50 mM Tris-HCl buffer (pH 8.0) and 50 µL of crude enzyme extract were incubated at 37 °C for 90 min. The reaction was then stopped by adding 50 µL of 1.5 M trichloroacetic acid (TCA, Sigma-Aldrich). To prepare the blank control, the crude extract was pre-incubated with TCA before adding the substrate to inhibit the enzymatic activity.
Centrifugation was performed on both test and control samples to discard protein precipitates, and the ammonia level in the supernatant was analyzed using the Nesslerization method. In a test tube, 100 µL of the clear supernatant was mixed with 700 µL of distilled water, and then 100 µL of Nessler’s reagent was added. The reaction mixture was then incubated at room temperature for 20 min. The yellow color indicated the presence of ammonia, and higher levels of ammonia resulted in the formation of dark orange to brown precipitates. The optical density at 480 nm was determined using a spectrophotometer (Hidex, Turku, Finland). Ammonia levels were measured using an ammonium sulfate-based standard curve. ASNase activity was expressed as the quantity of enzyme producing 1 µmol of ammonia per hour under the experimental conditions.

2.6. Influence of Incubation Time, Temperature, pH, and NaCl Levels on ASNase Activity

Optimization of incubation time for ASNase activity was carried out by incubating 50 µL of MABIK FU00000820 crude enzyme with 100 µL of 0.04 M L-asparagine at 37 °C for 15, 30, 45, 60, 75, 90, 105, and 120 min. To evaluate the effect of temperature, the reaction mixtures were incubated at various temperatures (4, 15, 25, 37, 50, and 65 °C) for 90 min. The influence of pH was assessed by incubating the enzyme with 0.04 M L-asparagine prepared in different buffer systems: acetate buffer (pH 4.0 and 5.5), sodium phosphate buffer (pH 7.0), and glycine-NaOH buffer (pH 8.5 and 10.0), at 37 °C for 90 min. The impact of salt concentration on enzyme activity was determined by supplementing the 0.04 M L-asparagine solution with 0, 2.5, 5, 10, and 15% (w/v) NaCl and incubating with the crude enzyme at 37 °C for 90 min. For preparation of the blank control, TCA was used to inactivate the crude enzyme extract prior to substrate addition. Using the Nesslerization method, ASNase activity was assessed in accordance with prior descriptions.

2.7. Thermostability of ASNase

The stability of ASNase at various temperatures was evaluated by preincubating the crude enzyme extract at various temperatures. The volume of 50 µL of the enzyme extract was incubated at 4 °C, 15 °C, 25 °C, 37 °C, 50 °C, and 65 °C for 2 h, then immediately cooled on ice. Following this step, 0.04 M L-asparagine was added to the enzyme and incubated at 37 °C for 90 min, after which ASNase activity was measured in both test and blank controls as outlined previously. Remaining activity was normalized against the control sample that had not been pre-incubated, which was set to 100%.

2.8. Data Analyses

Statistical evaluations were performed with GraphPad Prism 5.0, applying one-way analysis of variance (ANOVA) and, where appropriate, Tukey’s multiple comparison test or Student’s t-test. Experiments were generally conducted in triplicates unless noted otherwise.

3. Results

3.1. Examination of ASNase, GLNase, and Urease Activities in Marine Fungi

We investigated the activities of ASNase, GLNase, and urease in marine fungi by using a plate assay incorporating a pH indicator. The ASNase production by the marine fungal strain MABIK FU00000820 was confirmed by the formation of a pink zone around the colony when cultured in asparagine-containing medium at 25 °C for 7 d. No pink zones were observed on GLNase and urease assay plates, indicating the absence of detectable enzymatic activity (Figure 1). The findings imply that the marine isolate MABIK FU00000820 generates ASNase without detectable GLNase or urease activity.

3.2. Identification of Strain MABIK FU00000820

The strain MABIK FU00000820 was cultured on PDA at 25 °C for 7 d. The colony exhibited a circular, dense, and velvety texture with a grayish-olive coloration. The colony margins were even and slightly lighter in color. The reverse side of the colony was dark brown to blackish, consistent with the typical pigmentation of this species (Figure 2A). To evaluate the effect of salinity on radial growth, MABIK FU00000820 was cultured on PDA supplemented with various concentrations of NaCl. The strain exhibited better growth at lower NaCl concentrations, with a colony diameter of 4.30 ± 0.03 cm in the absence of NaCl. No growth was observed at NaCl concentrations 15% of above (w/v).
The genus Paraconiothyrium identification by phylogenetic analysis was performed using the ITS, D1/D2 domain, tub2, and tef1 genes [25,26]. For the molecular identification of the strain MABIK FU00000820, fragments of the ITS region (510 bp), D1/D2 domain (845 bp), tub2 gene (455 bp), and tef1 gene (945 bp) were analyzed. The corresponding sequences were deposited in GenBank under the accession numbers PV953296, PV953301, PV976142, and PV982388, respectively. A BLAST search using the ITS sequence indicated that MABIK FU00000820 shared 99.61% identity with P. cyclothyrioides strains UTHSC:DI16-279, B1P6-3, and CIRM-BRFM 3718, as well as Microsphaeropsis arundinis strain ZJ2-F. In the neighbor-joining phylogenetic tree, it clustered with these strains with 95% bootstrap support (Figure S1A). However, the phylogenetic analysis failed to resolve MABIK FU00000820 to the species level with certainty.
BLAST analysis of the D1/D2 domain sequence revealed that MABIK FU00000820 shared 100% identity with P. cyclothyrioides strains CBS 432.75 and UTHSC:DI16-252, P. maculicutis CBS 101461, P. marshiae BRIP 75785aT, and P. bishopiae BRIP 72437bT, and 99.64% identity with P. estuarinum CBS 109850T. In the neighbor-joining phylogenetic tree based on the D1/D2 region, MABIK FU00000820 clustered with these strains, supported by an 83% bootstrap value (Figure S1B). Similarly to the ITS-based phylogenetic analysis (Figure S1A), this analysis did not provide sufficient resolution to classify MABIK FU00000820 at the species level.
In contrast, the tub2 (BenA) and tef1 (EF) sequences of strain MABIK FU00000820 were clearly grouped with P. cyclothyrioides, supported by high bootstrap values of 99% and 90%, respectively. For the tub2 sequence, strain MABIK FU00000820 showed 100% identity with P. cyclothyrioides strains UTHSC:DI16-243 and CBS 972.95 (Figure 2B). BLAST analysis of the tef1 sequence revealed high similarity to P. cyclothyrioides strains UTHSC:DI16-346 (99.89%) and UTHSC:DI16-246 (99.79%) (Figure 2C). Taken together, the morphological characteristics and molecular data indicate that MABIK FU00000820 belongs to P. cyclothyrioides.

3.3. Influence of Cultivation Time on ASNase Activity Under Submerged Fermentation

To determine the optimal cultivation time for maximum ASNase activity, cultures previously grown in PDB were transferred to MCD broth and incubated for 18, 24, 48, and 72 h. Enzyme activity was quantitatively measured in mycelia collected at each time point. Maximum activity was observed at 48 h and 72 h, with no significant difference between them (p > 0.05). The activity at 48 h (0.35 units) was approximately 4.4-fold higher than that at 18 h (0.08 units) (Figure 3).

3.4. Influence of Incubation Time on ASNase Activity

The data in Figure 4 demonstrate the relationship between incubation time and ASNase activity, measured every 15 min over the 15–120 min range. The highest ASNase activity of MABIK FU00000820 was recorded at 90 min (0.31 units), after which the activity gradually declined with longer incubation times. No significant difference in ASNase activity was observed between 90 and 105 min (p > 0.05). However, the differences in activity between 75 and 90 min and between 75 and 120 min were statistically significant (p < 0.05).

3.5. Influence of Temperature on ASNase Activity

To examine the influence of temperature on ASNase activity in MABIK FU00000820, the crude enzyme extract was incubated with asparagine at 4, 15, 25, 37, 50, and 65 °C for 90 min. ASNase activity gradually increased with rising temperature, reaching a peak at 37 °C (Figure 5). There were no significant differences in ASNase activity among 37 °C and 50 °C (p > 0.05), while no activity was detected when incubated at 65 °C.

3.6. Influence of pH on ASNase Activity

The influence of pH on ASNase activity was tested under pH 4, 5.5, 7, 8.5 and 10. The ASNase activities at pH 4 and 5.5 were barely detectable, and the highest activity was recorded at pH 8.5 (0.29 units) (Figure 6). The maximum activity at pH 8.5 was significantly different at pH 7 and 10, respectively (p < 0.05).

3.7. Effect of NaCl Concentration on ASNase Activity

The influence of NaCl on ASNase activity was assessed at salt concentrations of 0, 2.5, 5, 10, and 15% (w/v). Maximum activity was detected at 0% NaCl (0.33 units), with a significant difference of 2.5% (0.14 units) (p < 0.05) (Figure 7). ASNase activity decreased progressively between 0% and 5% NaCl, with activity being nearly absent at concentrations of 5% or higher. The ASNase activity was nearly undetectable at NaCl concentrations between 5% and 15%, approaching zero.

3.8. Thermal Stability of MABIK FU00000820 ASNase

The temperature impact on the stability of ASNase showed maximum activity at 4, 15, and 25 °C, with no significant differences among them (p > 0.05). More than 77% of the remaining activity showed consistency with that of the untreated control (Figure 8) wihin this temperature range. There was no residual activity after pre-incubation at 37, 50, and 65 °C.

4. Discussion

The adverse effects and reduced efficacy of the currently available chemotherapeutic ASNases are largely attributed to their associated GLNase and urease activities [11]. To address this limitation, bacterial studies, particularly in E. coli and E. chrysanthemi, have employed molecular engineering to retain ASNase activity while minimizing GLNase activity. In these ASNases, substitution at defined amino acid residues induced conformational changes that improved substrate specificity [7,8]. As a result, glutamine turnover was much more severely impaired compared to asparagine hydrolysis. However, such structure-guided approaches have rarely been applied to fungal ASNases, and structural features of fungal ASNases remain largely unknown [11,12,13].
Fungi of the genus Paraconiothyrium are ascomycetous fungi that are globally distributed and associated with a broad range of hosts. They produce various secondary metabolites such as sesquiterpenes, diterpenes, polyketides, and aromatic compounds with antimicrobial, anticancer, and anti-inflammatory activities [27]. In particular, marine-derived Paraconiothyrium sp. VK-13 produces phenolic compounds with significant anti-inflammatory effects [28]. To date, P. cyclothyrioides has been primarily investigated for its morphological characteristics and pathogenicity in humans [29,30], and only limited information is available on its enzymatic properties and production of bioactive compounds. Although it has been isolated from halophytes inhabiting salt marshes, it is not frequently recovered from either terrestrial or marine environments in Korea [31].
During a plate assay-based screening for marine fungi producing ASNase lacking both GLNase and urease activities, strain MABIK FU00000820 was identified. The ASNase from MABIK FU00000820 exhibited maximum activity after 90–105 min of incubation, followed by a slight decline at 120 min. Such a time-dependent reduction in enzymatic activity has been documented in other microbial ASNases [23] and is generally attributed to factors including enzyme instability, product inhibition, substrate depletion, or pH fluctuations [32]. In the case of the MABIK FU00000820 ASNase, the reduced activity is possibly associated with its low thermal stability at 37 °C. The optimal temperature of this ASNase was slightly higher than those reported for filamentous fungi (30–45 °C), while the pH profile was comparable to other fungal ASNases [33,34,35,36].
This study employed an unpurified crude enzyme, making it difficult to directly compare its activity with that of commercial ASNase. It should be noted that crude enzyme preparations inherently contain various undesired impurities in addition to ASNase. When intracellular enzymes are extracted by ultrasonication, cytoplasmic components (proteins, enzymes, polysaccharides, nucleic acids, lipids, etc.), as well as disrupted cell wall fragments (glucans, chitin, glycoproteins, etc.), may be present [37,38].
Importantly, the absence of GLNase and urease activities in this strain highlights its potential value for therapeutic applications. Although its low thermal stability may restrict direct clinical use, stability could be improved through stabilizers or optimized reaction conditions [39,40]. Alternatively, the ASNase from this strain may also be applied at relatively low temperatures to reduce acrylamide formation in foods. ASNase is used not only as an anticancer therapeutic but also as a food additive to mitigate acrylamide generated during baking or frying [41]. Although the higher thermal stability of food-grade ASNases enhances their effectiveness in acrylamide reduction, it has been reported that treating dough with ASNase during the resting stage at 10–20 °C can decrease acrylamide formation in bread by 76–87% [42].
To ensure competitiveness with commercial ASNase, further steps are required, including enzyme purification, production optimization, validation of anticancer efficacy, and enhancement of enzyme stability. Moreover, while ultrasonication is suitable for small- to medium-scale production, large-scale recovery of interacellular ASNase may require alternative mechanical disruption strategies (bead mill, high-pressure homogenization) or more gentle chemical methods [43]. Therefore, the development of new extraction methods for intracellular ASNase of MABIK FU00000820 warrants further investigation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/applmicrobiol5030100/s1, Figure S1. The phylogenetic trees of a marine fungal strain MABIK FU00000820.

Author Contributions

W.-J.Y. and H.Y.L. conducted the experiments. W.-J.Y. and D.C. designed the experiments, analyzed the data, and wrote the manuscript. Y.M.K., S.S.B. and H.-J.H. contributed scientific insights to the experimental methods and discussion. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the National Marine Biodiversity Institute of Korea (MABIK) through its in-house research program (2025M00600).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are openly available in repositories. The names of repositories, strain ID, and accession numbers can be found below: https://doi.org/10.5281/zenodo.16777452 (accessed on 21 July 2025), https://www.mbris.kr/biobank/ (accessed on 21 July 2025), MABIK FU00000820 (strain identity); https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 21 July 2025), PV953296, PV953301, PV976142, and PV982388 (accession numbers).

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ALLAcute lymphoblastic leukemia
ASNaseL-Asparaginase
GLNaseL-Glutaminase
ITSThe internal transcribed spacer
LSUThe partial D1/D2 domain of a large subunit of rDNA
PDAPotato dextrose agar
PDBPotato dextrose broth
TCATrichloroacetic acid

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Applmicrobiol 05 00100 g001
Figure 1. Identification of a marine fungal isolate producing ASNase without GLNase and urease activity. The strain MABIK FU00000820 was inoculated onto MCD medium supplemented with asparagine, glutamine, urea, or NaNO3 (control). The cultures were inspected for the appearance of a pink zone after 5 days of incubation. ‘+’ and ‘−’ indicate the presence and absence of enzymatic activity for ASNase, GLNase, and urease, respectively.
Figure 1. Identification of a marine fungal isolate producing ASNase without GLNase and urease activity. The strain MABIK FU00000820 was inoculated onto MCD medium supplemented with asparagine, glutamine, urea, or NaNO3 (control). The cultures were inspected for the appearance of a pink zone after 5 days of incubation. ‘+’ and ‘−’ indicate the presence and absence of enzymatic activity for ASNase, GLNase, and urease, respectively.
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Figure 2. Identification of MABIK FU00000820. (A) MABIK FU00000820 was cultured on PDA containing various concentrations of NaCl at 25 °C for 7 d. (B,C) The neighbor-joining trees using sequences of the β-tubulin (BenA) and the translation elongation factor 1α (EF). The numbers at nodes indicate the percentage bootstrap values based on 1000 replications.
Figure 2. Identification of MABIK FU00000820. (A) MABIK FU00000820 was cultured on PDA containing various concentrations of NaCl at 25 °C for 7 d. (B,C) The neighbor-joining trees using sequences of the β-tubulin (BenA) and the translation elongation factor 1α (EF). The numbers at nodes indicate the percentage bootstrap values based on 1000 replications.
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Figure 3. ASNase activities at different cultivation times during submerged fermentation. Fungal cultures previously grown in PDB were transferred to MCD broth and incubated for 18, 24, 48, or 72 h. ASNase activity was assessed in mycelia collected at each time point. ‘*’ and ‘ns’ indicate differences that are statistically significant (p < 0.05) and non-significant (p > 0.05) following t-test.
Figure 3. ASNase activities at different cultivation times during submerged fermentation. Fungal cultures previously grown in PDB were transferred to MCD broth and incubated for 18, 24, 48, or 72 h. ASNase activity was assessed in mycelia collected at each time point. ‘*’ and ‘ns’ indicate differences that are statistically significant (p < 0.05) and non-significant (p > 0.05) following t-test.
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Figure 4. Effect of incubation time on the ASNase activity of MABIK FU00000820. The crude enzyme was reacted with the asparagine solution for varying incubation times. ASNase activity was measured at 15 min intervals from 15 to 120 min. ‘*’ and ‘ns’ indicate differences that are statistically significant (p < 0.05) and non-significant (p > 0.05) following t-test.
Figure 4. Effect of incubation time on the ASNase activity of MABIK FU00000820. The crude enzyme was reacted with the asparagine solution for varying incubation times. ASNase activity was measured at 15 min intervals from 15 to 120 min. ‘*’ and ‘ns’ indicate differences that are statistically significant (p < 0.05) and non-significant (p > 0.05) following t-test.
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Figure 5. Temperature influence on ASNase enzymatic activity of MABIK FU00000820. The crude enzyme preparation was mixed with asparagine solution and maintained at 4 °C, 15 °C, 25 °C, 37 °C, 50 °C, and 65 °C for 90 min. ‘ns’ indicates differences that are statistically non-significant (p > 0.05) following t-test.
Figure 5. Temperature influence on ASNase enzymatic activity of MABIK FU00000820. The crude enzyme preparation was mixed with asparagine solution and maintained at 4 °C, 15 °C, 25 °C, 37 °C, 50 °C, and 65 °C for 90 min. ‘ns’ indicates differences that are statistically non-significant (p > 0.05) following t-test.
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Figure 6. pH-dependent variation in ASNase activity of MABIK FU00000820. The crude enzyme was incubated with asparagine solutions prepared at pH 4, 5.5, 7, 8.5, and 10 at 37 °C for 90 min. ‘*’ indicates statistically significant differences (p < 0.05) following t-test.
Figure 6. pH-dependent variation in ASNase activity of MABIK FU00000820. The crude enzyme was incubated with asparagine solutions prepared at pH 4, 5.5, 7, 8.5, and 10 at 37 °C for 90 min. ‘*’ indicates statistically significant differences (p < 0.05) following t-test.
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Figure 7. ASNase activity of MABIK FU00000820 under varying NaCl concentrations. A crude enzyme was incubated with asparagine solutions containing NaCl at concentrations of 0%, 2.5%, 5%, 10%, and 15% (w/v) at 37 °C for 90 min. An asterisk (*) represents significant differences at p < 0.05 according to the t-test.
Figure 7. ASNase activity of MABIK FU00000820 under varying NaCl concentrations. A crude enzyme was incubated with asparagine solutions containing NaCl at concentrations of 0%, 2.5%, 5%, 10%, and 15% (w/v) at 37 °C for 90 min. An asterisk (*) represents significant differences at p < 0.05 according to the t-test.
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Figure 8. Thermal stability of MABIK FU00000820 ASNase. The crude enzyme was incubated at various temperatures (4–65 °C) for 1 h before conducting the activity assay. The remaining ASNase activity was then determined and expressed relative to the control sample without pre-incubation. ‘ns’ statistically non-significant differences (p > 0.05) following t-test.
Figure 8. Thermal stability of MABIK FU00000820 ASNase. The crude enzyme was incubated at various temperatures (4–65 °C) for 1 h before conducting the activity assay. The remaining ASNase activity was then determined and expressed relative to the control sample without pre-incubation. ‘ns’ statistically non-significant differences (p > 0.05) following t-test.
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MDPI and ACS Style

Yu, W.-J.; Lee, H.Y.; Kwon, Y.M.; Bae, S.S.; Hwang, H.-J.; Chung, D. Characterization of L-Asparaginase Free of L-Glutaminase and Urease Activity Produced by the Marine Paraconiothyrium cyclothyrioides Strain MABIK FU00000820. Appl. Microbiol. 2025, 5, 100. https://doi.org/10.3390/applmicrobiol5030100

AMA Style

Yu W-J, Lee HY, Kwon YM, Bae SS, Hwang H-J, Chung D. Characterization of L-Asparaginase Free of L-Glutaminase and Urease Activity Produced by the Marine Paraconiothyrium cyclothyrioides Strain MABIK FU00000820. Applied Microbiology. 2025; 5(3):100. https://doi.org/10.3390/applmicrobiol5030100

Chicago/Turabian Style

Yu, Woon-Jong, Ha Young Lee, Yong Min Kwon, Seung Seob Bae, Hyun-Ju Hwang, and Dawoon Chung. 2025. "Characterization of L-Asparaginase Free of L-Glutaminase and Urease Activity Produced by the Marine Paraconiothyrium cyclothyrioides Strain MABIK FU00000820" Applied Microbiology 5, no. 3: 100. https://doi.org/10.3390/applmicrobiol5030100

APA Style

Yu, W.-J., Lee, H. Y., Kwon, Y. M., Bae, S. S., Hwang, H.-J., & Chung, D. (2025). Characterization of L-Asparaginase Free of L-Glutaminase and Urease Activity Produced by the Marine Paraconiothyrium cyclothyrioides Strain MABIK FU00000820. Applied Microbiology, 5(3), 100. https://doi.org/10.3390/applmicrobiol5030100

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