Production of L-methioninase enzyme, investigation of its anticarcinogenic properties and usage areas in the food industry

In this study, L-methioninase was partially purified from Brevibacterium linens BL2. The specific activity of this enzyme was 3.055 units/mg. The cytotoxic properties of L-methioninase against Glioblastoma cells were evaluated. The IC50 values of U87MG and T98G glioblastoma cell lines were found to be 5.792 units/mL and 5.215 units/mL, respectively, using a neutral red viability assay after 24 h of incubation. L-methioninase also increased the cytotoxic effect of etoposide in the same cell lines when combined. This led to a notable decrease in the viability of U87MG cells, with only 24.35% remaining compared with the control group after 48 h of incubation. Similarly, the growth of T98G cells was inhibited by both L-methioninase and etoposide compared to the control group. Following After 48-hour phase of incubation, the cellular viability decreased by 37.74% compared to that of the untreated group. Notably, the cytotoxic effects of the L-methioninase enzyme produced by Brevibacterium linens on Mouse Embryonic Fibroblast (MEF) cells and Human Keratinocyte Cell Line (HaCaT) cells, however, were found to be lower than those of etoposide. The combined administration of L-methioninase and etoposide resulted in an overall decrease in HaCaT cell viability to 63.02% and MEF cell viability to 58.99%. The use of staining methods such as Giemsa staining, DAPI staining, and F-actin staining enabled the direct detection of alterations in both cell population and morphology. The co-administration of L-methioninase and etoposide showed notable effectiveness in suppressing cellular migration, as evidenced by the results of a wound healing experiment. In a clonogenic experiment, the administration of L-methioninase and etoposide led to a significant decrease in the number of colonies observed throughout the 21-day incubation period. According to the RT-qPCR results, L-methioninase caused a decrease in the expression of c-Myc and survivin genes, while simultaneously increasing the expression of Caspase-3. This study also investigated the effects of applying L-methioninase to cheddar cheese slurries on the production of volatile sulfur compounds and cheese microbiome alteration. The cheddar slurry samples were analyzed using next-generation sequencing and GC-MS analysis. On the first day of the experiment, the samples were mostly dominated by Lactococcus and Streptococcus strains. On the ninth day, a notable augmentation in microbial diversity was found in all samples, characterized by the presence of Limosilactobacillus, Lactobacillus, Enterococcus, and Lacticaseibacillus in considerable quantities across all samples. Lactococcus and Streptococcus were the predominant species. The introduction of L-methioninase and Brevibacterium linens BL2 led to the formation of volatile sulfur compounds, namely, Methanethiol and Dimethyl trisulfide. During the first stage of fermentation, the L-methioninase enzyme generated a quantity of 1490.2 ± 1.31 (µg/kg dry matter) of Methanethiol and 65.8 ± 0.04 (µg/kg dry matter) of Dimethyl trisulfide inside the cheddar slurry. The present study provides evidence supporting the possibility of L-methioninase as a promising option for food enzymes in cheddar cheese manufacturing, thus presenting substantial economic benefits.