Production and purification of methionine gamma-lyase from Iranian soil mulds: investigation of physicochemical properties and anticancer effects

Abstract

Cancer’s rising global incidence necessitates innovative therapies targeting the unique metabolic vulnerabilities of tumor cells. This study investigates microbial methionine gamma-lyase (MGL), an enzyme that depletes methionine, as a potential anticancer agent. Elevated methionine levels drive tumor progression by promoting S-adenosyl methionine (SAM) synthesis, leading to hypermethylation of tumor suppressor genes. By reducing methionine availability, MGL may inhibit SAM formation, preserving tumor suppressor function and hindering cancer growth. Three novel Iranian fungal strains—Penicillium flavigenum, Stagonosporopsis cucurbitacearum, and Penicillium allii—were identified as MGL producers through ITS sequencing. MGL was purified from P. flavigenum, and its biochemical properties, including stability, optimal pH, and temperature, were characterized, revealing high affinity for L-methionine and strong catalytic efficiency. Expression analysis of apoptotic genes (BCL-2 and caspase-3) demonstrated MGL’s role in enhancing apoptosis. In vitro assays confirmed its effectiveness against breast (MCF-7), liver (Hep G2), leukemia (MOLT-4), and glioblastoma (U87MG) cancer cell lines, with minimal toxicity to normal cells. This study underscores the therapeutic potential of fungal-derived MGL as a novel anticancer agent, highlighting its ability to modulate apoptotic pathways and providing a foundation for future clinical applications.

Introduction

With the rising incidence of cancer worldwide, there is a pressing need for new and effective therapeutic strategies for cancer treatment. One method that has gained attention in recent years is supportive therapy utilizing enzymes such as L-asparaginase [1, 2], L-glutaminase [3, 4], and L-methionase [5, 6]. Cancer cells require increased amounts of specific metabolic and nutritional substances, such as glucose and amino acids, particularly methionine, to support their rapid proliferation [7].

Methionine gamma-lyase is an enzyme that depletes methionine from the blood, leading to cell death in cancer cells by reducing their methionine intake. The antitumor mechanism of methionine gamma-lyase is primarily attributed to the depletion of molecules synthesized from methionine. High levels of methionine promote cancer growth as it serves as a precursor to S-adenosylmethionine (SAM). Elevated SAM levels can lead to hypermethylation of promoter regions in tumor suppressor genes, thus increasing the risk of tumor formation. By reducing methionine levels, the formation of S-adenosylmethionine is prevented, allowing tumor suppressor genes to remain unmethylated and thereby inhibiting cancer progression [8]. Additionally, methionine gamma-lyase breaks down methionine, which reduces polyamine synthesis. This reduction in polyamines is significant, as polyamines have been associated with increased cell proliferation and decreased apoptosis, both of which contribute to cancer risk. Recent research indicates that methionine gamma-lyase is effective in treating various types of cancer, often resulting in tumor shrinkage or disappearance. However, it is essential to note that methionine gamma-lyase is not used as a standalone treatment; instead, it is typically employed as a supportive or adjuvant therapy [9].

Laboratory studies, animal studies, and clinical trials conducted since the 1990 s have demonstrated that methionine restriction is an effective anticancer strategy. In vitro experiments using various cell lines have shown that recombinant methionine gamma-lyase is effective in treating colon cancer (Colo205 and SW620), skin cancer (A375), ovarian cancer (A2780), and breast cancer (MCF-7) [10]. Diets supplemented with methionine gamma-lyase significantly inhibited prostate tumor growth in mice. Additionally, levels of L-methionine, its conversion product L-cysteine, and the oxidative stress marker malondialdehyde were significantly reduced in vivo. These findings suggest a method for reducing L-methionine in foods through engineered methionine gamma-lyase, which effectively inhibits tumor growth in mice [11].

Methionine gamma-lyase has also been tested as a potent antiproliferative enzyme for human lung and colon cancers, glioblastoma, and neuroblastoma. Reports indicate that the growth of human tumors in vivo and in vitro (xenografted in nude mice) is inhibited compared to normal cells after treatment with polyethylene glycol conjugated with recombinant methionine gamma-lyase. Mouse models of colon, lung, and brain cancer have shown efficacy and synergy when treated with methionine gamma-lyase, either alone or in combination with chemotherapeutic agents such as cisplatin, 5-fluorouracil, and vincristine [12]. Moreover, methionine gamma-lyase has shown the ability to inhibit and treat prostate cancer, reducing or stabilizing prostate-specific tumor markers in patients with the disease [9]. In some cases, it has been effective in halting Lewis’s lung cancers, certain types of colon cancer, glioblastoma, neuroblastoma, and other cancer types [13].

Recent significant efforts in cancer research are focused on developing new forms of chemotherapy that leverage the clear distinction between cancer cells and normal cells, similar to the approach used with MGL [14, 15]. One distinct characteristic of certain types of cancer cells is their dependency on methionine [14]. Cancer cells have a firm reliance on methionine for their growth, and when they are deprived of it using MGL, their proliferation is significantly hindered. These cells cannot adapt to the loss of methionine, which makes them vulnerable. Because of this, using MGL in enzymotherapy is seen as a promising strategy for effectively targeting cancer cells [16]. Furthermore, neither healthy volunteers nor cancer patients have reported any adverse side effects from the oral administration of MGL [17].

Methionine gamma-lyase is found in nearly all organisms, including bacteria, fungi, protozoa, and plants, but is absent in mammals. Most bacteria, protozoa, and plants produce this enzyme intracellularly, while fungal sources tend to produce it extracellularly [18, 19]. Following initial studies, several researchers have concentrated on purifying, biochemically characterizing, and evaluating the therapeutic potential of methionine gamma-lyase as an anticancer agent against various human cancer cell lines [20–22]. Despite its wide applications, there have been few studies focusing specifically on eukaryotic (especially fungal) enzymes or comparing them with bacterial sources. In general, the therapeutic effects of bacterial enzymes are often associated with high immunogenicity, low substrate specificity, and potential toxicity to the kidneys and liver [7, 23, 24]. In contrast, these issues do not appear to be significant for fungal enzymes. Immunogenicity problems related to bacterial (prokaryotic) methionine gamma-lyase can be addressed through strategies such as polyethylene glycol-polyglycolylation or by using immunization techniques that combine T-cell epitope deletion with neutralizing drift. Additionally, fungal sources tend to produce higher quantities of the enzyme compared to bacterial sources [23].

This study primarily focuses on screening for L-methionine-producing mulds found in soil. It reports for the first time the three strains of bacteria, Penicillium flavigenum, Stagonosporopsis cucurbitacearum, and Penicillium allii, which are novel producers of L-methioninase. These strains may serve as promising alternative resources for cancer treatment. Additionally, we have purified methionine gamma-lyase from novel strains of Penicillium flavigenum and reviewed its pharmaceutical formulation. Furthermore, the anticancer efficacy of the purified enzyme should be evaluated against various cancer cell lines in comparison to regular cell lines. This study is also the first to investigate the impact of methionine gamma-lyase enzyme expression on the apoptotic genes BCL-2 and caspase-3 in both cancerous and healthy cells. This research aims to identify potential targeted cancer treatments and to understand the physicochemical properties of the enzyme for drug development. It also explores the effects and toxicity of the methionine gamma-lyase enzyme on the expression of apoptotic genes in both cancerous and healthy cells.

Materials and methods

Sample collection

Soil samples were collected from twenty different locations across Iran at a depth of 10 to 15 cm from the soil surface (to maintain some moisture) and transferred to sterile sampling containers.

Purification of Muld samples

After the incubation period, a small square piece of muld was cut from the edge of each muld colony, which had a distinct shape and color. Using a sterile loop, the samples were transferred along with the underlying agar to a Petri dish containing dextrose agar culture medium enriched with yeast extract and containing the antibiotic ampicillin, all while maintaining sterile conditions. These cultures were then incubated at 30 degrees Celsius for 7 to 10 days. After the growth period, the mulds were examined for uniformity in shape and color across the entire surface of the culture medium, and pure cultures were prepared from any impure mulds.

Production culture to induce L-methioninase production in mulds

A modified liquid Zapec-Dox culture medium was prepared using the ingredients listed in Table 1, and its pH was adjusted to 6. The prepared culture medium was sterilized through a 0.22 μm syringe filter. Subsequently, 15 ml of the sterilized medium was distributed into empty 50 ml Erlenmeyer flasks that had also been sterilized at 121 °C and 15 psi for 17 min. Purified mulds were cut from the slant culture into 0.4 cm x 0.4 cm pieces using a sterile loop and transferred into the Erlenmeyer flasks containing the culture medium, ensuring maintaining sterile conditions. The cultures were then incubated for 72 h in a shaking incubator set at 30 °C and 130 RPM.

Table 1.

Ingredients and concentrations needed to prepare the modified liquid Zapec-Dox culture medium

Substance	Concentration
Sucrose	3%
L-methionine	0.5%
Dipotassium hydrogen phosphate	0.1%
Magnesium sulfate	0.05%
Potassium chloride	0.05%
Ferric chloride	0.001%

Quantitative assay of L-methioninase production by mulds

After 72 h of incubation, 0.2 ml of the crude enzyme was extracted from the muld samples’ liquid culture medium, ensuring that the colonies growing in the medium remained undisturbed. This was added to a Falcon tube containing 1.8 ml of a 20 mM L-methionine solution in a 50 mM Tris-HCl buffer with a pH of 7.2. The reaction solution was then incubated for 60 min at 30℃. After incubation, 0.2 ml of 15% trichloroacetic acid was added to stop the enzymatic reaction. The reaction solution was centrifuged for 10 min at 4000 rpm to precipitate the mycelium and any other solid materials that may have been present. After centrifugation, 0.2 ml of the supernatant was removed and transferred to a microtube containing 1.6 ml of distilled water, followed by vortexing. Next, 0.2 ml of Nessler’s reagent was added to the microtube, which was immediately vortexed and incubated for 10 min at room temperature. The optical absorbance of the assay sample was measured immediately after the incubation period at a wavelength of 425 nm, compared to its corresponding blank sample.

Each assay sample had its own preparation for the blank sample. In the first step, 0.2 ml of crude enzyme was extracted from the muld culture medium for the assay sample. In comparison, an equal volume of crude enzyme was simultaneously added to a Falcon tube containing 1.8 ml of 50 mM Tris-HCl buffer without L-methionine. The remaining steps for the blank sample were performed in the same manner and concurrently with the assay sample.

Molecular characterization of Muld isolates using ITS-rDNA

The muld isolates that exhibited the highest activity of MGL were characterized molecularly using ITS-rDNA sequencing. For ITS-rDNA amplification, the primers ITS1 and ITS4 were utilized as forward and reverse primers, respectively. The merged ITS-rDNA amplified sequences were analyzed using the nucleotide BLAST (BLASTn) tool from the NCBI database. The results were evaluated based on similarity, total score, and coverage, ultimately identifying the genus and species of the muld in question. Additionally, the desired sequence and the top twenty sequences from the database displaying the highest similarity were recorded in FASTA format using MEGA software version 11.0.13. These sequences were then aligned with the ALIGN tool, employing the MUSCLE algorithm. Finally, a phylogenetic tree was constructed using the neighbor-joining method.

Protein determined

Protein levels were determined utilizing the Bradford method, using bovine serum albumin as the standard [25].

Purification of L-methioninase from Penicillium flavigenom

The culture medium underwent incubation for 72 h, followed by centrifugation for 10 min at a speed of 6,000 rpm. The resulting clear supernatant, evaluated for enzymatic activity, served as the crude enzyme preparation for subsequent purification steps. This research was conducted in three distinct stages to purify the L-methioninase enzyme.

The clear supernatant was subjected to thermal treatment at 55 °C and 60 °C for 20, 10, and 30 min, respectively. This step was designed to selectively denature unwanted proteins while preserving the enzymatic activity of L-methioninase. Following this treatment, the supernatant was rapidly cooled on ice for one hour to stabilize the enzyme, and the denatured proteins were subsequently removed through centrifugation at 5,000 rpm at 4 °C for 15 min.

In the second purification phase, the sample demonstrating L-methioninase activity underwent dialysis. It was introduced into a Sephadex C-50 ion exchange chromatography column (25 × 25 cm), previously equilibrated with a 25 mM potassium phosphate buffer at pH 7.2. The chromatography columns were washed with two bed volumes of the same phosphate buffer to elute unbound proteins. Bound proteins were eluted stepwise using a linear sodium chloride gradient from 1.0 to 0.1 M in the phosphate buffer at a flow rate of 70 ml/h. Collected fractions were analyzed for protein concentration at an absorbance of 280 nm using the Bradford assay, and both bound and unbound protein fractions were assessed for their L-methioninase activity. Active fractions were then combined and concentrated through subsequent dialysis and ultrafiltration steps [15].

In the final stage, to achieve greater purity, in addition to the previous steps, the concentrated protein samples were further analyzed using gel filtration chromatography on a Sephadex G-100 column (2 × 50 cm). The fractions were eluted with a 25 mM potassium phosphate buffer at pH 7.2, at a flow rate of 30 ml/h, with an initial sample volume of 5 ml. Protein concentration was again estimated by measuring the absorbance at 280 nm, while L-methioninase activity was evaluated for each fraction. Active fractions were subsequently concentrated through freeze-drying and stored at −80 °C for future use.

SDS-PAGE analysis assessed the purity of the fractions exhibiting the highest protein concentration and L-methioninase activity, comparing the results against standard marker proteins.

Biochemical characterizations of lmethioninase from Penicillium flavigenom

Thermal stability and the ideal temperature for the MGL

To evaluate the thermal stability of the methionine gamma-lyase enzyme, we took 1 ml of the enzyme solution and subjected it to temperatures of 4, 25, 45, 50, 60, 70, 80, 90, and 98 ℃ for periods of 30, 60, and 120 min. After each temperature exposure, we extracted 0.2 ml of the sample and conducted an enzyme assay, which was then compared to a control.

Furthermore, we determined the optimal temperature for the activity of methionine gamma-lyase produced by P. flavigenom. This was achieved by combining 0.2 ml of the enzyme solution with 1.8 ml of methionine substrate at various temperatures of 20, 30, 40, 50, 60, 70, 80, 90, and 100 ℃, and the Nessler detection method assessed enzyme activity and compared the results to the control.

The consistency of pH and the ideal pH for the MGL reaction

To assess the enzyme’s stability across different pH levels, we mixed 1 mL of the enzyme solution with 1 mL of phosphate buffer (or phosphate citrate) within a pH range of 3 to 8. The mixture was refrigerated at 4 ℃ for two hours before measuring the enzyme activity. We also compared these results against a control.

Additionally, to ascertain the optimal pH for the reaction involving MGL, we combined 0.2 mL of the enzyme with 1.8 mL of 0.1 M phosphate-buffered saline (PBS) at varying pH levels of 5, 6, and 7, and the culture medium pH of 6.5. After introducing methionine into the mixture, we evaluated and compared the enzyme activity with the control using Nessler’s method.

The influence of various inhibitors and activators on the function of the enzyme MGL

To assess the activators and inhibitors, a range of ions at a concentration of 3 mM—including K+, Na+, Zn2+, Mg2+, Ba2+, Ca2+, Hg2+, Cu2+, Al3+, and Fe3+—were employed alongside harmful amino acid analogs such as sodium dodecyl sulfate (SDS), hydroxylamine, hydrogen peroxide, ethanol, methanol, guanidine thiocyanate, and 2-mercaptoethanol at a concentration of 10 mM. These substances were stored in a refrigerator at 4 °C for 2 h. Subsequently, the enzyme activity was measured using the standard procedure and compared to the control.

NaCl at a concentration of 2 mM, which enhanced the MGL activity, was further tested at concentrations ranging from 1 to 10 mM.

The preference of the MGL enzyme for various substrates

Enzymes demonstrate specific functions, but because of the structural similarities among different amino acids, they can interact with one another’s substrates. To evaluate the affinity of methionine gamma-lyase from Penicillium flavigenom for various amino acids, we combined 0.2 mL of the enzyme solution with 1.8 mL of substrate for each of the following amino acids: L-methionine, L-cysteine, homocysteine, L-ornithine, L-arginine, L-glycine, L-tyrosine, and L-asparagine. We assessed the enzyme activity using the standard method and compared the outcomes to a control. This study used purified L-methioninase to calculate kinetic parameters, such as the Michaelis-Menten constant (Km) and maximum velocity (Vmax).

In vitro evaluation of the anticancer properties of purified MGL derived from Penicillium flavigenom on transformed cell lines

Investigating the toxicity and lethality of the enzyme on various cell lines

A variety of cell lines, including MCF7 (breast cancer), MOLT-4 (leukemia), Hep G2 (liver cancer), U87MG (human glioblastoma), and a regular human melanocyte cell line (HFB4), were obtained from the Pasteur Institute in Iran. All cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin, maintained at 37 °C in a 5% CO2 atmosphere.

The viability of cancer cells was assessed in the presence of MGL derived from P. flavigenom using the MTT assay. Suspensions of MCF7, MOLT-4, Hep G2, and U87MG cells (1.2 × 10^4 cells/mL) were plated in 96-well plates, with 200 µL of cell suspension added to each well and cultured for 24 h. Following this, the cells were treated with various concentrations of purified MGL (1000, 500, 250, 120, 60, 30, 15, 8, and 0 µg/mL) for an additional 24 h.

Following the treatment, the MTT assay was conducted under dark conditions by incubating for 2 h at 37 °C with MTT (5 mg/mL in distilled water; 20 µL per well). Formazan crystals were solubilized using DMSO (100 µL), and the optical density of the 96-well plate was measured at 570 nm with an automated plate reader. For each 96-well plate, six vehicle controls containing either media or 0.5% DMSO were included. The MTT assay assessed the number of viable cells after 24 h of incubation. The viability and inhibition ratios of MGL were calculated using the following equations:

Investigation of apoptotic and anti-apoptotic gene expression in cancer cell lines following treatment with methionine gamma-lyase

This study assessed the expression levels of apoptotic genes BCL-2 and caspase 3 using Real-Time PCR. Initially, RNA was extracted from cells treated with a concentration of IC 50 MGL and from the control group (untreated) using an RNA extraction kit (Qiagen, USA). The concentration of the extracted RNA was then measured with a Nanodrop device (Scientific Fisher Thermo, USA). Subsequently, cDNA synthesis was conducted using a kit (Takara, Japan). The reaction mixture, comprised of 5 µl of buffer and 1.8 µl of RNA, was diluted to a final volume of 10 µl with double-distilled water. The temperature-time program for cDNA synthesis included primer binding for 5 min at a specified temperature, followed by cDNA synthesis at 42 °C for 60 min, and inactivation of the reverse transcription enzyme at 85 °C. The GAPDH gene was utilized as the internal control (housekeeping gene). The primers for the Bcl-2, caspase 3, and GAPDH genes are detailed in Table 2. The Real-Time PCR reaction mixture consisted of 5 µl of Master PCR Green Syber, 0.4 µl of reverse primers for each gene, 1 µl of cDNA, and 0.4 µl of Dye Reference, brought to a final volume of 10 µl with double-distilled water. The PCR was performed using the One Step Qia quant 96 plex (USA) with the following temperature conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 34 s. This investigation focused on the expression of apoptotic and anti-apoptotic genes in cancer cell lines following treatment with methionine gamma-lyase.

Table 2.

Sequence of primers of Bcl-2, caspase 3, and GAPDH genes

gene	Primer 5′ to 3′	Refrence
Bcl-2	F: 5′-ACCTACCCAGCCTCCGTTAT-3′ R: 5′-GAACTGGGGGAGGATTGTGG-3′	(65)
Caspase 3	F: 5′-GCTGGATGCCGTCTAGAGTC-3′ R: 5′-ATGTGTGGATGATGCTGCCA-3′
GAPDH	F: 5′-GAA GGT GAA GGT CGG AGT C-3′ R: 5′-GAA GAT GGT GAT GGG ATT TC-3′

Statistical analysis

The tests in this study were conducted in triplicate, and statistical analysis was performed using GraphPad Prism software version 10. The cytotoxicity and gene expression data were evaluated using one-way analysis of variance (ANOVA). A p-value of less than 0.05 was deemed statistically significant, and results were presented as mean ± standard deviation (SD). Furthermore, the difference in threshold cycles between the tested samples (cells treated with a concentration of IC 50 MGL) and the control samples (cells untreated with the combination mentioned above) was calculated using the real-time PCR method and the ΔΔCt formula. The gene ratio of the target gene to the reference gene (GAPDH) was determined using the formula 2^−ΔΔCt.

Result

Screening and characterization of MGL-producing Muld isolates

A dilution series was prepared from soil samples collected using the described method and cultured on Petri dishes containing dextrose agar enriched with yeast extract and antibiotic ampicillin. The cultures were incubated at 30 °C for 3 to 7 days. The muld samples were quantitatively assessed for L-methioninase production using the Nessler method. Some samples that did not exhibit growth in the culture medium were excluded from the assay.

During this process, three indicator species associated with the production of the methionine gamma-lyase enzyme were identified. Each of these species underwent molecular evaluation using the ITS-rDNA method, allowing for the determination of their genus and species. The three muld species identified as producing the enzyme in this study were Penicillium flavigenum, Stagonosporopsis cucurbitacearum, and Penicillium allii. A phylogenetic tree was constructed for each species, and the findings were subsequently registered in the NCBI database (Fig. 1).

Fig. 1.

ITS-rDNA phylogenetic tree: (A) Penicillium flavigenum isolate MA01, (B) Stagonosporopsis cucurbitacearum isolate MA01, (C) Penicillium allii isolate MA01

Purification of MGL from Penicillium flavigenum

Among the three muld species that produce methionine gamma-lyase enzyme, Penicillium flavigenum, which had the highest crude enzyme production in the culture medium extract, was selected for enzyme purification and further studies.

To isolate the active extracellular crude L-methioninase from the Penicillium flavigenum, the supernatant was harvested using a cooling centrifuge at 6000×g for 10 min. The resulting crude enzyme exhibited an activity of 23.13 U/mL and a protein content of 2.30 mg/mL. This crude enzyme was subsequently purified to apparent homogeneity through three purification steps: heat treatment, a Sephadex C-50 ion exchange column, and a Sephadex G100 column. The heat treatment was implemented to eliminate denatured proteins from the supernatant, which resulted in a slight decrease in enzyme activity to 17.64 U/mL and a 52.2% reduction in protein content. The purification fold achieved was 1.59, with a specific activity of 16.03 U/mg and a recovery yield of 76.26%.

Additionally, this step effectively reduced the protein content in the precipitation fraction, which resulted in the highest enzyme recovery. Further purification steps were essential to attain a greater purification fold. Data obtained indicated that the purification of L-methioninase on CM Sephadex C-50 revealed that fractions 6 to 15 exhibited enzyme activity with a purification fold of 2.96 and a 54.04% yield. Enzymatic activity was also detected in fractions 15 to 18, which displayed higher protein content. The active fractions were pooled together, increasing specific activity (68.66 U/mg) and a purification fold of 6.83 (Table 3).

Table 3.

Purification steps, purification folds, and recovery yields of L-methioninase from P.flavigenom

Sample	Total protein (mg/ml)	Total activity(U/ml)	Specific activity (U/mg)	Enzyme recovery (%)	Purification fold
Curd MGL	2.30	23.13	10.05	100	1
Heated treatment	1.10	17.64	16.03	76.26	1.59
CM Sephadex C-50	0.42	12.50	29.76	54.04	2.96
Sephadex G100 (Gel filtration)	0.12	8.24	68.66	35.62	6.83

SDS-PAGE was conducted following the concentration of the active fractions of Sephadex G100 to verify enzyme purity and molecular weight. The results displayed a single band at an approximate molecular mass of 62 kDa, indicating the presence of two identical subunits, which confirmed the dimeric structure of L-methioninase from P. flavigenom (Fig. 2).

Fig. 2.

SDS-PAGE analysis of different stages during the purification of MGL from P. flavigenom Line 1: marker, Line 2:After Sephadex G-100,Line 3: After CM Sephadex C-50,Line 4:After Heating

Optimization of reaction parameters to increase the activity of L-methioninase from P. flavigenom

Impact of incubation temperature and buffer pH on MGL activity and stability

The stability of the methionine gamma-lyase enzyme was tested at temperatures of 4, 25, 45, 50, 60, 70, 80, 90, and 98 ℃ for 30, 60, and 120 min without substrate presence. Subsequently, the enzyme activity was measured using Nessler’s reagent. The enzyme’s stability at temperatures below 45 ℃remained unchanged over 2 h. However, at 50 ℃, a 30% decline in enzyme activity was observed after 120 min. At 60 ℃, the enzyme activity decreased by 40% of its initial level after 60 min, and it reached half of its initial activity after 80 min. At temperatures exceeding 90 ℃, the enzyme exhibited no activity after 120 min. Specifically, at 98 ℃—the boiling point of water at the testing location—no enzyme activity was detected after just 60 min. (Fig. 3A).

Fig. 3.

Effect of pH and temperature on P.falvigenom L-methioninase: (A) thermal stability of methionine gamma-lyase from P.falvigenom; (B) effect of temperature on P.falvigenom L-methioninase activity; (C) effect of pH on P.falvigenom L-methioninase activity; (D) effect of pH on P.falvigenom L-methioninase stability

Enzyme activity was assessed at various temperatures, 20, 30, 40, 50, 60, 70, 80, 90, and 100 ℃. The highest enzyme activity was observed at temperatures between 30 and 37 ℃. At 25 ℃, considered ambient temperature, enzyme activity was nearly equivalent to that at 50 ℃. The lowest enzyme activity was recorded at 100 ℃ (Fig. 3B). To evaluate the enzyme’s stability at different pH levels, the enzyme was incubated without substrate at pH values ranging from 3 to 11 for 60 min. The enzyme demonstrated the highest and most stable activity at pH levels of 5 to 7. It proved to be more resistant in acidic conditions, experiencing a significant decline in stability as the pH became more alkaline. The lowest enzyme activity was noted at pH 11 (Fig. 3C). Additionally, we examined enzyme activity across the same pH range. In the acidic range, the lowest activity occurred at pH 3, while the highest activity was found at pH 7, where enzyme activity reached 100%. The acidic environment had minimal impact on enzyme activity; even at pH 3, it retained 60% of its activity. In contrast, the enzyme was more sensitive to alkaline conditions as activity approached zero at pH 11 (Fig. 3D).

Influence of metal ions and buffer additives on the activity of purified MGL

Our experiments found that copper sulfate had the most substantial inhibitory effect on the enzyme methionine gamma-lyase, followed by iron sulfate. We observed no increase in enzyme activity when incubated with these ions. Notably, methionine gamma-lyase requires no metal coenzymes and functions independently (Table 4).

Table 4.

Effect of some metal ions and chemical reagents on P.flavigenom L-methioninase

Metal ions (2 mM)	Relative activity (%)	Chemical reagent(10mM)	Relative activity(%)
control	-	Hydroxylamine	31
KCl	81	Guanyldine thiocyanate	40
NaCl	95	2- mercaptoethanol	14
ZnCl2	82	H2O2	20
MgSO4	30	SDS	48
BaCl2	31	EDTA	100
CaCl2	89	Ethanol	98
HgCl2	62	Methanol	100
CuSO4	25
AlCl3	91
FeCl3	90
FeSo4	28

Various chemical reagents were assessed for their inhibitory effects on the methionine gamma-lyase enzyme. Among the reagents tested, 2-mercaptoethanol and hydrogen peroxide showed the most significant inhibitory impact, with over 80% inhibition achieved. Hydroxylamine and guanidine thiocyanate also exhibited notable inhibitory effects, exceeding 60% (Table 4). The methionine gamma-lyase enzyme from P. flavigenom is particularly promising for pharmaceuticals and biotechnology due to its resilience to ethanol and methanol.

Chlorine ions also decreased enzyme activity, with the most significant reductions observed for barium chloride and mercury chloride. To further investigate, we examined the effects of different concentrations of sodium chloride, ranging from 2 to 10 mM, on the enzyme. We found that sodium chloride concentrations up to 4 mM did not impact enzyme activity, but higher concentrations decreased activity. (Fig. 4A).

Fig. 4.

Determination of: (A) Effect on the activity of purified MGL by different consternation of NaCl; (B) substrate specificity; (C) Km and Vmax; (D) reaction time

Determination of substrate specificity, km, and vmax, and half-life of the purified MGL

This study was conducted to assess the specificity. Purified L-methioninase was tested with various substrates. Equal amounts (20 mM) of specific substrates, including L-methionine, L-cysteine, homocysteine, L-ornithine, L-arginine, L-glycine, L-tyrosine, and L-asparagine, were added individually to the reaction mixture and incubated under optimal assay conditions. The results, presented as relative activities in Fig. 4(B), demonstrated that the enzyme displayed the highest affinity for L-methionine, which served as the standard substrate. Furthermore, cysteine exhibited a degradation level of 80% in comparison to the activity of L-methionine. The activity of the enzyme toward homocysteine and L-ornithine was found to be less than 60% and 40%, respectively, compared to its activity with L-methionine. Studies on the substrate specificity of P. flavigenom L-methioninase showed that this enzyme exhibits different activity levels for various amino acids, with a significant preference for L-methionine. Additionally, L-cysteine and homocysteine serve as effective substrates.

The kinetic parameters for purified methionine gamma-lyase derived from P. flavigenom were ascertained through the application of a Lineweaver-Burk plot, plotting 1/V against 1/[S]. The values obtained for the Michaelis constant (Km) and the maximum velocity (Vmax) of the enzyme were found to be 19.25 mM and 0.8131 U/mL/min, respectively (Fig. 4C). The enzyme exhibited maximal activity at substrate concentrations of 60, 70, 80, 90, 100, and 120 mM for L-methionine. This suggests that the enzyme’s active site became saturated with the substrate at concentrations above 50 mM. The relationship between substrate concentrations and the reaction velocity demonstrated a hyperbolic pattern rather than a sigmoidal one, indicating that this enzyme may be classified as allosteric. The apparent Km value of methionine gamma-lyase indicates a high affinity for its substrate, highlighting its potential as an efficient antitumor agent. Furthermore, the catalytic efficiency of fungal L-methioninase was superior to that of the bacterial variants, as evidenced by the Km values for L-methionine.

The reaction time was systematically varied from 5 to 35 min to investigate its effect on enzyme activity. The highest activity of MGL was observed after 20 min of incubation. Following this, a gradual decline in activity was noted. The half-life (t1/2) of purified methionine gamma-lyase from P. flavigenom is 503.32 min (approximately 8.38 h) (Fig. 4D).

Impact of the purified methionine gamma-lyase derived from P. flavigenome on transformed cell lines

Impact of purified methionine gamma-lyase derived from P. flavigenom on both cancerous and normal cell lines in vitro

Different concentrations of purified methionine gamma-lyase from P. flavigenome, specifically 1000, 500, 250, 120, 60, 30, 15, 8, and 0 µg/ml, were tested for their ability to inhibit the growth of transformed cells, including MCF-7, Hep G-2, MOLT-4, and U87MG. The results showed that L-methioninase has a strong capacity to combat cancer cells. IC50 values for various cancer cell lines were determined to be over 47.63 µg/ml for breast cancer cells (MCF-7), 249 µg/ml for liver cancer cells (HepG-2), 257 µg/ml for leukemia cells (MOLT-4), and 267 µg/ml for human glioblastoma cells (U87MG). These results indicate that the purified methionine gamma-lyase from P. flavigenome is effective in destroying cancerous cells, as demonstrated by the MTT assay and analyzed using GraphPad Prism 10.2.3. In contrast, the L-methioninase enzyme did not affect the growth of normal melanocytes (HFB4 cells), even at concentrations significantly higher than the IC50, which was set at 1000 µg/ml. Methionine gamma-lyase is considerably less toxic to normal HFB4 melanocyte cells than to cancer cells. (Fig. 5)

Fig. 5.

Cell viability of transformed cell lines after 24 h treatment with purified MGL from P.falvigenom; (A) MCF-7; (B) Hep G-2;(C) U87MG and (D)MOLT-4;(E) Cytotoxicity of L-methioninase from P.falvigenom on the regular cell (HFB4)

In this study, the most significant effect of methionine gamma-lyase, purified from P. flavigenom, on cancer cell lines was observed in the human glioblastoma cell line MCF-7.

Expression analysis of Caspase-3 and BCL-2 genes in cancerous and normal cell lines in vitro under the influence of methionine gamma-lyase

This study examined the expression profiles of the caspase-3 and BCL-2 genes in various cancer cell lines treated with the IC50 concentration of methionine gamma-lyase derived from P. flavigenom over 24 h. Melting curve analysis was utilized to confirm the accuracy of the gene amplification. The results demonstrated that the GAPDH, caspase-3, and BCL-2 genes were amplified successfully. Notably, the expression of the caspase-3 gene exhibited a significant increase compared to the reference gene GAPDH across all cancer cell lines subjected to methionine gamma-lyase treatment after 24 h. In contrast, the expression ratio of the BCL-2 gene relative to the GAPDH reference gene showed a significant decrease in all cancer cell lines treated with methionine gamma-lyase during the same time frame (p < 0.05) (Fig. 6).

Fig. 6.

Expressions of BCL-2 and Caspase-3 in the various cell lines after 24 h

The MCF-7 cell line exhibited the most significant increase in caspase 3 expression, along with a reduced expression of Bcl-2, compared to the control group. In contrast, the regular HFB4 cell line showed no changes in the expression of either the caspase-3 or BCL-2 genes compared to the control group.

Discussion

Fungi represent a highly diverse group of organisms, ranging from yeasts and mulds to larger fungi. Most fungal species spend part of their life cycle in the soil or direct contact with it [26]. Mulds, in particular, exhibit a wide variety of enzymatic activities, making them potential candidates for industrial enzyme production [27]. Various microorganisms, including mulds, have been reported to produce L-methioninase, a therapeutic enzyme. However, there is still significant potential for further research to isolate Indigenous mulds from soils in different regions of the world to identify the highest producers of industrial enzyme applications [28]. Soils in different regions harbor unique microbial populations. Soil samples were collected from 20 distinct locations across Iran to enhance the diversity of the isolation process. Collecting samples from various locations leads to the identification of different strains, even when isolating mulds of the same genus and species. This variance is particularly significant, as previous studies on the isolation of L-methioninase-producing mulds from soil were conducted in countries like Egypt and India rather than in Iran [28, 29]. A dilution series was prepared using soil samples, similar to methods described in previous articles, to reduce density and facilitate purification [29]. In this study, the optimal dilution for the soil samples was found to be 1,600 times, eliminating the need for successive re-cultivations that could alter the enzymatic activity of the muld in the laboratory environment. The diluted samples were then cultured in a medium containing antibiotics. Ultimately, 65 mulds were purified and stored based on their morphological differences observed in the soil sample cultures. The production process was conducted using a modified Zapec-Dox culture medium and carried out in a submerged environment. In this process, the amino acid L-methionine was used as a nitrogen source instead of sodium nitrate to enhance enzyme production. The production period lasted 72 h, which allowed samples with faster growth and production to excel in the quantitative assay compared to those that grew slowly and yielded later. This shorter production time helped the superior samples become more competitive against bacterial producers, which typically have a production cycle of only 24 h. There are several methods for quantitatively measuring L-methioninase activity, depending on which products of L-methionine degradation are being analyzed. In this study, we used a quantitative method involving Nessler’s reagent, where ammonia produced from the enzymatic reaction of L-methioninase is measured. The three indicator mulds were samples C2, K4, and S2, which exhibited enzyme activities of 23.13 U/ml, 11.45 U/ml, and 11.4 U/ml, respectively.

The identification of fungi has traditionally relied on their phenotypic and physiological characteristics. However, the distinctive features of fungi can complicate identification and classification based on morphology. Therefore, molecular methods can provide a more accurate determination of taxonomy. In this study, the ITS region of the genomic DNA from various mulds was sequenced and analyzed using the NCBI database, leading to the identification of the genera and species of mulds that produce L-methioninase.

The results revealed that the C2 muld was identified as Penicillium flavigenum isolate MA01, the K4 muld as Stagonosporopsis cucurbitacearum isolate MA01, and the S2 muld as Penicillium allii isolate MA01. The production of the enzyme L-methioninase has been documented in several species of the genus Penicillium, including Penicillium digitatum, Penicillium notatum, Penicillium cassiculum, Penicillium citrinum, and Penicillium oxalicum [28, 30, 31]. However, this study reports the production of L-methioninase from Penicillium flavigenum and Penicillium alli for the first time. Additionally, the production of L-methioninase from the genus Stagonosporopsis, specifically the species Stagonosporopsis cucurbitaceum, is also reported for the first time in this research.

In this study, the primary enzyme was purified through a three-step process that involved heat treatment, Sephadex C50, and Sephadex C100 gel filtration. Upon completion of the purification, the enzyme exhibited a specific activity of 68.66 U/ml and a recovery rate of 35.62%. This purification strategy, characterized by its minimal steps and impressive enzyme yield, has proven to be both practical and economically beneficial for the industry [25]. The heating process was crucial in reducing unwanted proteins by 47.8%, as demonstrated in the purification profile (Table 3). In a previous study focused on the purification of L-methioninase from Citrobacter freundii, a heat treatment at 60 °C was applied, resulting in a 21% recovery using an initial DEAE-cellulose column [31]. Furthermore, heat treatment of L-methioninase derived from Streptomyces DMMHM4 achieved an even greater purity, eliminating over 50% of the protein content. In another investigation involving A. fumigatus, a similar two-step approach—consisting of heat treatment followed by Sephadex chromatography—was employed, successfully removing approximately 45% of the heat-denatured proteins while preserving 81.2% of the enzyme’s activity [32, 33]. Also, other studies have indicated that the species Trichoderma harzianum exhibits the highest enzyme activity reported among mulds. This activity was achieved following several stages of enzyme purification, resulting in the following specific activities: crude enzyme 6.66 U/ml)10.4U/mg), ammonium sulfate precipitation 39.47 U/ml (12.24 U/mg), ion exchange 56.47 U/ml (41.03 U/mg), and gel filtration chromatography 88.83 U/ml (74.4 U/mg) [33]. Another study reported enzyme production by Candida tropicalis, which achieved a crude specific enzyme activity of 1.5 U/mg, which increased to 64.78U/mg after several purification stages [34] Additionally, the enzymatic activity of Aspergillus flavipes was measured at 22.65 U/ml (43.4 U/mg) in a soybean solid culture medium and 19.55 U/ml (40 U/mg) in a chicken feather solid culture medium. Notably, with the alkaline pretreatment of the chicken feather medium, this enzymatic activity increased to 71 (U/mg) [35].

The molecular weight of methionine gamma-lyase isolated from P. flavigenom was determined to be 62 kDa using SDS-PAGE. Notably, the molecular weights of L-methioninase purified from various other microorganisms show significant variation. For instance, A. favipes has been reported to have a molecular weight of 47 kDa [36], while T. harzianum exhibits a molecular weight of 48 kDa [37] Similarly, A. ustus and C. tropicalis both have molecular weights of 46 kDa [22, 38]., whereas (A) thaliana has a molecular weight of 212 kDa [39], (B) linens has 170 kDa [40], and A. flavipes shows 180 kDa [30]. These discrepancies in molecular weight across different microorganisms may arise from the presence or absence of denaturing agents during the purification process. Additionally, the enzyme purification method utilized in this study diverges from those employed in previous research.

The storage and utilization of the drug product should adhere to specific temperature guidelines to preserve its stability and efficacy. It is advisable to maintain the product at a temperature of 25 °C to ensure optimal stability and sustained activity. Enzyme activity peaks at temperatures between 30 °C and 40 °C, although this optimum range can differ depending on the microbial source. For instance, Aspergillus flavipes thrives at 20–40 °C, Candida tropicalis operates best at 45–55 °C, Streptomyces sp. functions optimally at 70 °C, and Bacterium leucovorum is most effective at 25–30 °C. Notably, enzyme activity tends to diminish when temperatures exceed the specified optimum range [40, 41]. Research on bacteria and fungi indicates that the methionine gamma-lyase enzyme derived from fungal sources exhibits better resistance to temperature variations and can maintain functionality at elevated temperatures compared to its bacterial counterpart.

In this context, methionine gamma-lyase (MGL) from P. flavigenom exhibits maximum activity at pH values between 6 and 7. The fungal enzyme demonstrates more significant activity in acidic environments compared to its bacterial counterpart. The fungal enzyme has an impressive ability to work well even when the pH levels fluctuate. It shows excellent resilience in acidic conditions, keeping about 60% of its activity even at pH 3. However, it doesn’t perform as well in alkaline environments, where its activity drops significantly. This lower activity in alkaline conditions might be linked to the naturally acidic environment in which these mulds thrive within the soil. The MGL enzyme remains active within a pH range of 4.0 to 8.0. Previous research indicates that the purified MGL from Aspergillus flavipes has optimal activity at pH 7 to 8, aligning with pH ranges between 6.8 and 8.0 [42].

In contrast, the purified MGL from C. tropicalis and Streptomyces sp. demonstrates optimal activities at pH 6.5 and 6.0, respectively. The purified MGL from Candida tropicalis achieved peak activity at pH 6.5 in sodium citrate and potassium phosphate buffers, with consistent performance across pH ranges from 5.5 to 7.0. Also, the purified MGL from Streptomyces sp. showed maximum activity in acetate buffer (50 mM) at pH 6.0. In comparison, in potassium phosphate buffer (50 mM), optimal MGL activity was observed at pH levels ranging from 6.5 to 8.0 [15, 24].

The stability of methionine gamma-lyase from P. flavigenom was examined across various pH buffers. It was found that the enzyme exhibited significant catalytic stability over a broad pH range, specifically between 4.0 and 9.0. Notably, the enzyme’s activity decreased considerably under both acidic and alkaline conditions. Similarly, investigations into the pH stability of L-methioninase from Aspergillus ustus, A. flavipes, and Trichoderma harzianum indicated that optimal activity was present within the pH range of 6.5 to 8.5 [22, 37, 38, 43]. Additionally, a study on L-methioninase from Candida tropicalis revealed that the enzyme retained activity at pH levels from 5.5 to 7.5, with only a slight decline noted at pH 8.0. The most significant drop-in activity was observed in the acidic range, particularly at pH 5.0 [24].

Based on the results obtained, none of the metal ions had a positive effect on the activity of the methionine gamma-lyase enzyme derived from P. flavigenum. Additionally, this enzyme is resistant to ethanol and methanol, which makes the enzyme from this muld a promising candidate for pharmaceutical and biotechnological applications. Previous studies indicate that methionine-gamma-lyase produced by various microorganisms exhibits enhanced enzyme activity in the presence of specific metal ions and chemical reagents, such as Cu2+, Mg2+, Co2+, and Mn2+. For instance, studies involving Aspergillus flavipes [24] and Candida tropicalis [44] revealed that Na+, Ni2+, and Mg2 + could increase enzyme activity. However, most studies have reported no significant increase in enzyme activity due to metal ions and chemical reagents, aligning with our findings. These investigations have shown that Cu2+, Fe2+, and Hg2 + inhibit methionine-gamma-lyase activity in Aspergillus flavipes and Streptomyces sp [45, 46]. Additionally, Ca2+, Mg2+, Zn2+, and Cd2 + exhibited a minor inhibitory effect on methionine-gamma-lyase produced by T. harzianum [37]. Methionine-gamma-lyase from A. fumigatus was notably inhibited by Hg2+, Cu2+, Li2+, and Mn2+, while only slight inhibition was observed with Ni2+, Fe3+, and Cr3+ [47]. Other studies have explored the effects of chemical reagents on methionine-gamma-lyase; for example, enzymes from K. oxytoca demonstrated increased activity with EDTA and Tween-80. In contrast, reagents such as SDS, β-mercaptoethanol, DTT, and PMSF were found to decrease enzyme activity in Streptomyces sp. and Aspergillus flavipes [45, 46]. Furthermore, EDTA and PMSF reduced enzyme activity in Streptomyces variabilis [48], while PMSF and EDTA also diminished enzyme activity, and iodoacetate inhibited activity in methionine-gamma-lyase from Candida tropicalis [44].

The findings from this study showed that methionine gamma-lyase obtained from P. flavigenom exhibited a strong affinity for L-methionine. After L-methionine, it also showed a considerable affinity for L-cysteine. Several researchers noted that it had a clear preference for both the primary substrate and its related compounds, L-cysteine and homocysteine. Studies have indicated that L-methioninases from A. flavipes and T. harzianum display different levels of catalytic activity towards various amino acids. They showed a high specificity for L-methionine, followed by L-cystine and L-cysteine [36, 37]. Conversely, A. stus L-methioninase exhibited the most excellent activity against the standard substrate while demonstrating reduced activity towards other substrates. The hydrolytic function of L-methioninase in cleaving C-S and C-O bonds is predominantly seen in enzymes sourced from bacteria [49–52], protozoal [53, 54], and fungi rather than in those interacting with C-C bonds (23.45). Additionally, research on the substrate specificity of L-methioninase from C. tropicalis revealed differing relative activities towards various sulfur-containing amino acids [38].

The Km and Vmax values for purified methionine gamma-lyase from P. flavigenom, utilizing L-methionine as the substrate, were found to be 19.25 mM and 0.8131 U/mL/min, respectively. These findings suggest that methionine gamma-lyase has a high affinity for its substrate (L-met), which supports its potential use as an anti-cancer therapeutic agent. The Km value provides insight into how effectively an enzyme binds to its substrate, reflecting the enzyme’s affinity and can be influenced by various factors such as temperature and pH. A higher Km signifies a lower affinity, indicating that more substrate is required for the enzyme to achieve half of its maximum activity. Conversely, Vmax is not a constant value for an enzyme; it can vary based on the enzyme concentration in a reaction, meaning it does not always represent the fundamental traits of the enzyme. Nonetheless, Vmax remains an essential indicator of an enzyme’s catalytic efficiency, offering information on how many substrate molecules can be converted into a product by a single enzyme molecule within a minute [14, 41, 49, 50]. Our research showed the peak enzyme activity at the 20-minute time point, aligning with earlier studies that reported methionine gamma-lyases from Arabidopsis and Candida tropicalis exhibited their highest activity at the same duration [39, 55].

The results from the cancer cell lines used in this study indicate that the examined enzyme significantly affects these cells, prompting them to undergo apoptosis. Additionally, past research has observed that cancer cells exhibit a specific metabolic defect related to the essential amino acid methionine, which may be connected to their increased dependence on tumor growth [41, 51]. The anticancer efficacy of the purified enzyme, L-methioninase, was assessed in vitro through a series of experiments involving various cancerous and normal cell lines. The results demonstrated a pronounced effectiveness of L-methioninase in targeting cancer cell lines while exhibiting a minimal cytotoxic impact on normal cells. This selective activity suggests a promising therapeutic potential for L-methioninase in cancer treatment, warranting further investigation into its mechanisms and clinical applications.

Various researchers have studied the effects of this enzyme from different sources on cancer cell lines and found similar results. The most notable impact of purified methionine gamma-lyase from P. flavigenom was observed in the human breast cancer cell line MCF-7. A prior study involving L-methioninase from T. harzianum demonstrated comparable anticancer activity against Hep2-G cell lines, followed by MCF-7 cell lines, with inhibition ratios of 47.62% and 33.84%, respectively, at a concentration of 1 U/mL [36]. Purified L-methioninase derived from A. favipes demonstrated considerable anti proliferative activity against five tested cell lines. The enzyme exhibited a concentration-dependent efficacy against various cancer cells, including lung, breast, liver, prostate, and colon [56]. These findings are consistent with previous studies [47, 52, 53]. Another investigation assessed the use of recombinant L-methioninase to inhibit the growth of MOLT-4 leukemia cells, yielding results similar to those observed with methionine gamma-lyase from P. flavigenom. This study confirmed its capability to impede the growth of cancer cells under laboratory conditions. The U87MG cell line has been evaluated in earlier research involving L-methioninase from K. oxytoca and a variant employing RBC for MGL transport, both of which corroborate the effects of L-methioninase in this context [53, 54] Extensive studies in this field indicate that L-methioninase is effective against most human cancer cell lines, primarily due to cancer cells’ dependency on methionine for survival [21, 57, 58].

The findings revealed that L-methioninase demonstrated a much higher IC50 of over 1000 µg/ml, indicating that its toxicity towards normal melanocyte HFB4 cells is minimal compared to its effects on cancer cells. Normal cells possess L-methionine synthase, which enables them to produce L-methionine from homocysteine (Hcy) by utilizing methyltetrahydrofolate and betaine as methyl group donors. Hcy, a non-standard amino acid, has a structure resembling L-methionine but lacks a methyl group. Research has shown that tumor cell lines dependent on L-methionine synthase generally exhibit lower enzyme levels compared to their normal cell equivalents [44, 47, 48].

For the first time in this study, we assessed the expression of apoptotic genes, specifically Caspase 3 and BCL-2, in cancer cell lines treated with methionine gamma-lyase derived from P. flavigenom. Following treatment with this enzyme, we observed an increase in the expression of Caspase 3, while the expression of BCL-2, which is an anti-apoptotic factor, decreased. This change suggests an increased rate of apoptosis in cancer cells exposed to methionine gamma-lyase from P. flavigenom. The MCF-7 cell line exhibited the most significant rise in Caspase 3 expression and a decrease in BCL-2expression, which aligns with the results obtained from the MTT assay for the purified methionine gamma-lyase from P. flavigenom.For the first time in this study, we assessed the expression of apoptotic genes, specifically Caspase 3 and BCL-2, in cancer cell lines treated with methionine gamma-lyase derived from P. flavigenom. Following treatment with this enzyme, we observed an increase in the expression of Caspase 3, while the expression of BCL-2, which is an anti-apoptotic factor, decreased. This change suggests an increased apoptosis rate in cancer cells exposed to methionine gamma-lyase from P. flavigenom. The MCF-7 cell line exhibited the most significant rise in Caspase 3 expression and a decrease in BCL-2expression, which aligns with the results obtained from the MTT assay for the purified methionine gamma-lyase from P. flavigenom.

In multicellular organisms, the careful regulation of programmed cell death, known as apoptosis, is just as essential as cell growth. Although the mechanisms controlling cell death are not yet fully understood, caspases play a crucial role in apoptosis. They are activated in a specific sequence that leads to distinct apoptotic changes. Despite their essential function in this process, the activation of caspases is influenced by a variety of other factors [59].

Moreover, the BCL-2 family plays a crucial role in triggering caspases and deciding a cell’s destiny—whether it continues to live or undergoes apoptosis. The members of the BCL-2 family mainly interact with the mitochondria, reacting to circumstances that cause irreversible cellular harm. Earlier molecular research has suggested a negative correlation between the levels of Caspase 3 and BCL-2 expression [60].

Ultimately, Increased expression of caspase-3 and decreased expression of BCL-2in cancer cells treated with methionine gamma-lyase likely activate the mitochondrial apoptotic pathway [60–62].

Conclusion

This study sheds light on the exciting potential of methionine gamma-lyase (MGL) from the P. flavigenom as a promising new treatment option for human cancer. The researchers successfully purified and characterized this enzyme, achieving a notable 6.83-fold enhancement in activity along with a respectable 35.62% recovery rate, all while confirming that it has a molecular weight of 62 kDa. These findings suggest that MGL could be effectively delivered using nanoparticle systems designed for the precise targeting of cancer cells.

The enzyme shows impressive stability, remaining active up to 45 °C, with peak performance between 30 and 40 °C and at a pH of 6 to 7. This robustness makes MGL a strong candidate for various therapeutic applications. What’s particularly noteworthy is its significant anticancer activity against multiple cancer cell lines, including MCF-7 (breast cancer), Hep G-2 (liver cancer), MOLT-4 (leukemia), and U87MG (brain cancer), while leaving healthy cells unharmed.

The way MGL works is quite fascinating. It triggers a process called apoptosis—or programmed cell death—via the mitochondrial pathway. This process aims to enhance the activity of the caspase-3 gene while diminishing the activity of the BCL-2gene. Both genes play crucial roles in regulating cellular life and death. Gaining insight into how this mechanism operates not only clarifies the role of the enzyme but also indicates potential therapeutic advantages.

Additionally, since MGL is derived from a eukaryotic source, it’s likely to be more compatible with the human immune system, which reduces the chances of adverse reactions that sometimes occur with non-native proteins. The findings of this research have significant implications, indicating that MGL may serve as a pivotal agent in oncological therapies while also potentially contributing to the management of various other health conditions. These include the regulation of elevated homocysteine levels, cardiovascular diseases, neurodegenerative disorders such as Alzheimer’s, as well as metabolic concerns including obesity and the aging process.

This study highlights the therapeutic potential of fungal-derived methionine gamma-lyase (MGL) as a promising anticancer agent, particularly its ability to modulate apoptotic pathways and inhibit tumor growth. However, several limitations should be acknowledged to pave the way for future research. Firstly, while in vitro experiments demonstrated significant anticancer effects, the enzyme’s performance in vivo remains unexplored, necessitating preclinical animal studies to evaluate its efficacy, pharmacokinetics, and safety. Secondly, the stability and activity of MGL under physiological conditions require further investigation to ensure its suitability for clinical applications. Additionally, the potential immunogenicity or adverse effects of fungal-derived MGL in human subjects must be rigorously assessed. Lastly, while this study focused on a limited set of cancer cell lines, expanding the scope to other cancer types and exploring combination therapies with existing treatments could enhance the therapeutic applicability of MGL.

Future research should address these challenges, focusing on optimizing MGL production, improving its stability, and exploring its broader anticancer potential in vivo. Investigations into its molecular mechanisms of action, particularly in modulating apoptotic pathways, could provide deeper insights into its therapeutic benefits and support its transition to clinical use.

In summary, these findings open the door to exciting new research possibilities, not just for cancer therapy but for a range of other conditions. The study highlights the promising role of methionine gamma-lyase in molecular biology and oncology, encouraging scientists further to explore its therapeutic applications in a clinical setting.

Author contributions

M.N. wrote the main manuscript text and conducted experiments related to the purification and examination of the physicochemical properties of the methionine gamma-lyase enzyme and examined the effect of this enzyme on various cancer cell lines and prepared all tables and all figures except Figure 1.A.A. Isolation of molds from the soil, identification of enzyme-producing molds, and determination of the genus and species of molds by molecular methods prepared Figure 1.M.M. Determining the study methodology and team supervisor and reviewed the manuscript.S.F. Determining the study methodology and team advisor and reviewed the manuscript.

Funding

The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.