L-methioninase producing bacterial strain

Methylobacterium sp. JUBTK33, isolated from the soil of a fenugreek farmland in Sethuvalli, Tamil Nadu, India, and identified as the highest L-methioninase producer among 75 microbial isolates in our previous study report [6], was selected for the current study.

Chemicals

The chemicals utilized in the current investigation were L-methionine, Pyridoxal-5-phosphate, and L-ascorbic acid (sourced from HIMEDIA, India), Coomassie Brilliant Blue, Bovine Serum Albumin (BSA), MEM media, fetal bovine serum, trypsin, MTT dye (all procured from HIMEDIA, India), di-sodium monohydrogen phosphate (Na2HPO4), monopotassium dihydrogen phosphate (KH2PO4), di-potassium monohydrogen phosphate (K2HPO4), sodium chloride (NaCl), magnesium sulfate (MgSO4), calcium chloride (CaCl2), D-Glucose, phenol red, agar, trichloroacetic acid, and Nessler’s reagent (also known as potassium tetraiodomercurate (II)), all of which meet analytical grade standards (sourced from Merck, Mumbai, India).

L-methioninase assay and specific activity of the enzyme

To assess L-methioninase activity in the culture supernatants containing the crude enzymes, we employed a modified version of the standard Nesslerization method, following the established protocol with minor changes [10]. The assay procedure involved the incubation of 1 mL of a 1% L-methionine substrate, prepared in a phosphate buffer (50 mmol, pH 7.0), with the addition of 0.1 mL of pyridoxal phosphate (100 μmol) and 1 mL of the crude enzyme extract. These components were thoroughly mixed and allowed to incubate for 60 min at 37 °C. To halt the enzyme-catalyzed reaction, 0.1 mL of trichloroacetic acid (1.5 mol) was added. The ammonia released during the reaction was quantified by introducing 0.5 mL of Nessler’s reagent (comprising HgCl2, KI, and NaOH), and the resulting color change was measured with a UV–VIS spectrophotometer (Shimadzu, Japan) at 480 nm against an enzyme blank. The calculated enzyme activity was expressed as μmol/min/mL. The assays were conducted three times, and the average value was taken into consideration. Specific activity was calculated by dividing the L-methioninase activity by the total protein content and expressed as unit activity per milligram (U/mg) of protein [6].

Total protein estimation

Lowry’s method was used to determine the total protein content of the extracted enzyme, with bovine serum albumin serving as the standard [11].

Enzyme purification

L-methioninase purification was performed using the acetone precipitation method with a 1:2 ratio of sample to acetone [12]. The precipitate was collected and subsequently subjected to partial purification.

Anion exchange DEAE column chromatography

The acetone-precipitated enzyme was passed through a DEAE cellulose column (Bio-Rad, UK) that had been pre-equilibrated with 0.05 M potassium phosphate buffer at pH 6.5. NaCl (0.12 M) was used as the elution buffer for the enzyme. Fifty 1 mL/min fractions were collected in separate tubes, and enzyme activity was determined using Nessler’s method. Active fractions were collected, pooled, and further purified using a Sephadex G-200 column [13].

Sephadex G-200 purification

The active fractions from the DEAE column were passed through a Sephadex G-200 column. The column was equilibrated with 0.05 M potassium phosphate buffer at pH 6.5. The enzyme was eluted at a rate of 1 mL/min with 0.12 M NaCl, and 50 fractions were collected and checked for enzyme activity. Active fractions were pooled and lyophilized for further use [13].

Molecular weight determination and activity staining

The purity of semi-purified (SPMet) and Sephadex G-200 column-purified (SG purified) enzymes was assessed, and their molecular weights were determined using 10% SDS–polyacrylamide gel electrophoresis following standard protocols [14]. The activity of the purified enzyme was confirmed by activity staining on modified starch agar plates supplemented with phenol red (0.07% w/v) as the indicator. The development of pink color will indicate the release of ammonia by the action of L-methioninase on methionine.

Evaluation of physicochemical parameters on SG purified L-methioninase enzyme activityThe effect of temperature on L-methioninase activity

By incubating the reaction mixture in a water bath for 1 h at various temperatures (20, 30, 40, 50, 60, and 70 °C), the effect of temperature on L-methioninase enzyme was determined [13]. The enzyme activity was determined after it was pre-incubated as per previously given methodology.

The effect of pH on L-methioninase activity

To determine the optimum pH for L-methioninase activity, acetate buffer (pH 5–6), phosphate buffer (pH 6.5–7.5), and Tris–HCl buffer (pH 8–9) were used. Enzymatic activity was determined following Nessler’s method, after incubating each reaction mixture at 37 °C for 30 min [13].

The effect of metal ions on L-methioninase activity

The effect of various metal ions, including Fe++, Li+, Na+, Ni++, Mn++, and K+, on L-methioninase activity was determined by pre-incubating the purified L-methioninase enzyme with metal ions at concentrations of 1 and 5 mM in 50 mM phosphate buffer at 37 °C for 1 h. After pre-incubation, enzyme activity was assessed following Nessler’s method [13].

Kinetics of column-purified L-methioninase enzyme

We utilized a conventional procedure to investigate how varying substrate concentrations (45 mM, 90 mM, 135 mM, 180 mM, 225 mM, and 280 mM) impacted the activity of L-methioninase. From the Lineweaver–Burk plot, we determined the enzymatic kinetic parameters, namely the Michaelis–Menten constant (Km) and the maximum velocity (Vmax) [13].

Cytotoxicity assay and safety assessment (Toxicity study)

The cytotoxicity of crude, semi-purified (SPMet), and Sephadex G (SG) purified L-methioninase (L-MET) alone and in combination with tamoxifen citrate (TAM) was tested on HepG2 (Liver cancer), MCF-7 (Breast cancer), and HeLa cells (Cervical cancer), with Doxorubicin (DOX) serving as the positive control (standard), using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay as per the standard methodology [15]. The cancer cells were incubated for 24 h, 48 h, and 96 h with different doses (10, 50, 100, 200, 400 µg/mL) of the enzyme in combination with 5 µg/mL and 10 µg/mL of TAM. After the treatment duration cytotoxicity was assessed by treating with MTT dye for 3 h, and later taking the optical density at 540 nm in an ELISA reader (Lisa Plus, Aspan, India). The safety and toxicity of the enzyme were assessed on Healthy human embryonic kidney (HEK-293) cell lines through the MTT assay.

Statistical analysis

In this present study, every experiment was conducted in triplicate, and the outcomes were expressed as Mean ± standard error. The experimental data were analyzed using one-way/two-way ANOVA within the Graph Pad Prism version 6 software. To assess the mean differences, the Dunnett’s multiple range test, also known as the Dunnett’s multiple comparison test, was employed. Statistical significance was considered at a p value less than 0.05.