Screening, isolation, and fermentation optimization of indigenous Streptomyces spp. for enhanced methioninase production

 Corresponding author: Pratik Yadav. Email: Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Screening, isolation, and fermentation optimization of indigenous Streptomyces spp. for enhanced methioninase production Pratik Yadav 1, *, Rakesh Mehta 2 and Ragini Gothalwal 3 1 Department of Biotechnology, Barkatullah University, Bhopal, M.P. India. 2 Department of Botany and Biotechnology. Govt., MGM College Itarsi. M.P. India. 3 Department of Biotechnology, Barkatullah University, Bhopal, M.P. India. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 266-275 Publication history: Received on 20 December 2024; revised on 10 February 2025; accepted on 13 February 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.21.2.0131 Abstract This preliminary study focuses on the isolation and characterization of indigenous Streptomyces spp. from the Upper Lake of Bhopal, Madhya Pradesh, India, with an emphasis on their potential to produce L-methioninase, an enzyme with significant therapeutic applications in cancer treatment. Sample collection was conducted from five locations around the lake, followed by isolation on starch casein agar and characterization through morphological and biochemical tests. The screening identified isolates capable of methioninase production, with two isolates (S2 and S3) demonstrating significant enzyme activity. The study further optimized liquid state fermentation conditions to enhance enzyme production, evaluating the effects of various carbon sources, pH levels, and temperature. Results indicated that sucrose was the most effective carbon source, yielding the highest enzyme activity (S2: 21.236 U/mL; S3: 23.243 U/mL) at neutral pH (7) and 30°C. The findings underscore the influence of environmental factors on microbial diversity and enzymatic potential in nutrient-rich ecosystems like the Upper Lake. This research contributes valuable insights into bioprospecting for indigenous microbial strains with promising enzymatic properties, highlighting the potential for developing effective microbial-based therapies for cancer treatment through optimized L-methioninase production. The results lay the groundwork for future studies aimed at large-scale enzyme production for therapeutic applications, advancing the field of enzyme therapy in oncology. Keywords: L-Methioninase; Streptomyces Spp; Liquid State Fermentation; Cancer Therapy; Microbial Diversity 1. Introduction Methioninase is a crucial enzyme with potential therapeutic applications, particularly in cancer treatment, due to its ability to degrade methionine, an essential amino acid that supports tumor growth and metastasis (Zhang et al., 2017). The increasing interest in methioninase for use in enzyme therapy targeting methionine-dependent cancers has spurred research into its production (Tan et al., 2019). Streptomyces spp. are renowned producers of secondary metabolites, including various enzymes, making them prime candidates for exploration in diverse ecological niches (Shirling & Gottlieb, 1966). This study emphasizes the screening and isolation of indigenous Streptomyces species from different locations of the Upper Lake of Bhopal, Madhya Pradesh, India. The Upper Lake, recognized as one of the largest artificial lakes in Asia, presents a unique aquatic ecosystem enriched with microbial diversity due to its varying environmental conditions, making it an ideal source for novel actinobacterial strains (Rai et al., 2021). Previous research has demonstrated that soil and sediment samples from aquatic environments harbor Streptomyces species with diverse enzymatic capabilities, including methioninase production (El-Naggar & Eldin, 2020). World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 266-275 267 The primary aim of this research is to isolate and screen methioninase-producing Streptomyces spp. from the Upper Lake of Bhopal, followed by the optimization of fermentation conditions to enhance enzyme production. Screening and isolation are essential steps in this study as they allow for the identification of strains with high methioninase activity, which is crucial for potential applications in cancer therapy. Additionally, optimizing fermentation parameters—such as pH, temperature, and nutrient sources—will be pivotal in maximizing methioninase production. This work aims to contribute to the growing field of bioprospecting for indigenous microbial strains with valuable enzymatic properties, ultimately leading to the development of effective microbial-based therapies for cancer treatment. 2. Materials and Methods 2.1. Isolation & Screening of Streptomyces Species 2.1.1. Sample Collection Marshy soil mixed water samples were collected from five locations around the Upper Lake of Bhopal, India, during the sampling period from August 2022 to July 2023. Pre-sterilized glass bottles were used for sample collection, and precautions were taken to avoid contamination. Each sample was assigned a code and stored at 4-8°C in the laboratory. The different sampling points are mentioned in table 1. Table 1 The different points of soil mixed water sampling around Upper Lake of Bhopal S.No. Sampling Points Sample Description Sample Code 1. Van Vihar National Park Turbid and muddy water S1 2. Boat Club Turbid and muddy water S2 3. Kamla Park Turbid and muddy water S3 4. Kaliasot Dam Turbid and muddy water S4 5. Bairagarh Turbid and muddy water S5 2.1.2. Isolation of Streptomyces Species Starch casein agar (M801 HiMedia) was used for isolating Streptomyces spp. from the samples, followed by sub-culturing on inorganic salt starch agar (ISSA) for pure cultures. Samples were serially diluted and inoculated onto agar plates using the spread plate method, followed by incubation at 30°C for 8-10 days. The composition of the media is mentioned in table 2. Colonies were enumerated using a digital colony counter, and pure cultures were prepared through repeated sub-culturing (Shirling & Gottlieb, 1966). Table 2 Composition of starch casein agar media (M801 HiMedia) S.No. Ingredients Quantity in Grams/Litre 1. Soluble starch 10.00 2. Casein (Vitamin Free) 0.30 3. KNO3 2.00 4. MgSO4.7H2O 0.05 5. K2HPO4 2.00 6. NaCl 2.00 7. CaCO3 0.02 8. FeSO4.7H2O 0.01 9. Agar 18.00 10. Distilled water 1000 ml All ingredients homogenised in per litre distilled water then sterilized and poured in sterile plates World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 266-275 268 2.1.3. Preliminary Characterization The initial identification of the isolates was conducted through the examination of colony morphology and Gram staining. Subsequently, biochemical characterization was performed to further identify methioninase-producing strains according to Aneja (2003) which includes indole production, methyl red, Voges-Proskauer, Simmons citrate, catalase, gelatin hydrolysis, starch hydrolysis, and urease tests. 2.2. Confirmation & Quantitative analysis of Methioninase The indigenous Streptomyces spp. isolates were screened for methioninase production by assessing methionine degradation on starch agar plates containing phenol red. Positive isolates were further confirmed by observing pink coloration around colonies on modified starch agar. Quantitative analysis of methioninase production involved liquidstate fermentation using modified starch-nitrate medium, incubated at 28°C on a rotary shaker for 72 hours. The cellfree supernatant was collected via centrifugation. L-methioninase activity was quantified using Nessler’s method, with optimization of fermentation parameters such as pH, temperature, and carbon source evaluated based on previously established protocols (Patel et al., 2019; El-Naggar et al., 2020; Zhang et al., 2021). 3. Results and Discussion 3.1. Enumeration of target microbial species The CFU count from various soil samples diluted at 10–7 (see table 3) indicates substantial microbial diversity, with Kamla Park (348 CFU) exhibiting the highest microbial presence, followed by Kaliasot Dam (199 CFU), and Bairagarh (104 CFU). These data suggest a higher prevalence of potential methioninase-producing Streptomyces spp. in environments with elevated organic matter, such as park areas according to present work. This aligns with studies showing that nutrient-rich soils support the growth of Streptomyces spp. for anticancer enzyme production (Xu et al., 2020a). Table 3 Culture response on primary culture plates S.No. Master Plate Code Sample Dilution Used CFU Count 1. A Van Vihar National Park 15 2. B Boat Club 36 3. C Kamla Park 348 4. D Kaliasot Dam 199 5. E Bairagarh 104 3.2. Morphological and Biochemical characterization When selected colonies from master culture subjected to pure culture, almost all the Streptomyces spp. isolates share almost similar morphological features on culture conditions including leathery, tough, and have a dry, chalky appearance. However, the colour of the colonies of Streptomyces spp. isolates slightly varies from creamy white to greyish to pinkish which are also mentioned in table 4 The isolates when observed under microscope showed either branched mycelium or filamentous and exhibit a characteristic branching pattern with septa (Kumar and Singh (2020). The biochemical characterization of Streptomyces isolates as mentioned in table 5 shows a diversity of metabolic capabilities. All isolates are catalase-positive and capable of citrate utilization and starch hydrolysis, indicating robust metabolic activity. However, only isolates S1 and S4 tested positive for methyl red reduction, highlighting differences in fermentation pathways. The indole test was negative across all isolates, confirming their inability to produce tryptophanase. These findings are consistent with prior studies on Streptomyces metabolic diversity (Bibb et al., 2018a). World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 266-275 269 Table 4 Pure culture prepared on culture plates S.No. Isolate Code Sample Source Culture Description 1. S1 A White smooth flat colony 2. S2 A White filamentous colony 3. S3 A Greyish fimbriated mucoid colony 4. S4 B White filamentous colony 5. S5 B Greyish fimbriated mucoid colony Table 5 Outcomes Biochemical characteristics of indigenous methioninase enzyme producing Streptomyces spp. isolates obtained in the present study S.No. Isolates Results of Tests Conducted Gram’s Staining Cell Shape Catalase Indole production Methyl Red reduction VogesProskauer Citrate utilization Starch Hydrolysis Gelatinase test Urease test 1. S1 +ve F +ve –ve +ve +ve +ve +ve +ve –ve 2. S2 –ve F +ve –ve –ve –ve +ve +ve +ve +ve 3. S3 –ve F +ve –ve –ve –ve +ve +ve +ve +ve 4. S4 +ve F +ve –ve +ve –ve +ve +ve +ve +ve 5. S5 –ve F +ve –ve –ve –ve +ve +ve –ve –ve 3.3. Confirmation & Quantitative analysis of Methioninase According to table 6 shows that among the five Streptomyces spp. isolates, only isolates S2 and S3 exhibited methioninase production activity, while isolates S1, S4, and S5 did not produce the enzyme. This indicates variability in methioninase production potential, which may depend on genetic or environmental factors influencing enzyme synthesis. Table 6 Response of pure indigenous Streptomyces spp. isolates for methioninase production activity S.No. Isolate Code Methioninase production activity 1. S1 –Ve 2. S2 +Ve 3. S3 +Ve 4. S4 –Ve 5. S5 –Ve The outcomes of liquid state fermentation activity for L-methioninase production at different parameters including effect of different carbohydrate, pH and temperature conditions are mentioned in table 8, Table 9 and Table 10 respectively, in terms of release of ammonical content which is calculated with the help of standard curve plot of ammonia table 7 and Figure 1. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 266-275 270 Table 7 Ammonia as standard concentration vs absorbance at 425 nm to plot standard curve for estimation of ammonical content in samples using Nessler’s method for calculation of L-methioninase units in fermentum. S.No. Concentration of Ammonia in µl/ml Absorbance 1. 50 1.322 2. 25 0.741 3. 12.5 0.228 4. 6.25 0.102 5. 3.125 0.019 Instrument Used: Single beam visible range digital microprocessed spectrophotometer from Electronic India model EI-2305. Figure 1 Standard Plot for known concentration of Ammonia Standard at 425 nm. The Graph is obtained from Excel 2013 linear regression function The results from Tables 8, 9, 10, and 11 show the effects of different carbohydrates, pH, and temperature conditions on L-methioninase production by Streptomyces spp. isolates during liquid state fermentation (LSF). The use of sucrose as a carbon source yielded the highest enzyme production for both isolates (S2: 21.236 U/mL; S3: 23.243 U/mL), followed by lactose (S2: 19.251 U/mL; S3: 22.245 U/mL), indicating that these carbon sources most effectively supported Lmethioninase production. Mannitol and maltose were less efficient, with maltose showing the lowest enzyme production, especially for isolate S2 (12.225 U/mL). Regarding pH, the optimal value was pH 7 for both isolates (S2: 25.424 U/mL; S3: 21.758 U/mL), consistent with previous studies highlighting that neutral pH levels enhance microbial enzymatic activity. Lower pH values (pH 4 and pH 5) resulted in significantly reduced enzyme production, as acidic environments may inhibit Streptomyces metabolic functions. For temperature, 30°C proved to be the optimal condition for methioninase production (S2: 14.436 U/mL; S3: 23.844 U/mL), aligning with typical mesophilic growth patterns of Streptomyces species. Lower and higher temperatures reduced enzyme activity, with 40°C showing a significant drop in production, likely due to enzyme denaturation or decreased microbial growth rates. Overall, the optimized parameters for methioninase production include the use of sucrose as the carbon source, pH 7, and 30°C as the temperature, as detailed in Table 11. These findings are consistent with research by Veeranagouda et al. (2015a), which demonstrated that carbohydrate source, pH, and temperature significantly influence microbial enzyme production. Furthermore, Qureshi and Zubair (2020) highlighted the critical role of these parameters in enhancing enzyme yield among various Streptomyces species, emphasizing the importance of selecting the right growth World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 266-275 271 conditions for optimal enzyme activity. Figures 2 to 5 illustrate the graphical representation of the impact of different carbon sources, pH, and temperature on L-methioninase production by two Streptomyces isolates, with optimal production achieved using sucrose, pH 7, and 30°C. Additionally, Sharma and Soni (2019) reviewed the applications of microbial enzymes in various industries, reinforcing the significance of optimizing environmental factors for maximizing enzyme production in microbial systems. Table 8 Effect of different carbohydrates on production of L-methioninase during LSF S.No. Isolates Units of L-methioninase Production due to use of different carbon sources at a concentration of 30gm per litre in LSF medium Sucrose Dextrose Mannitol Lactose Maltose 1. S2 21.236 18.225 15.324 19.251 12.225 2. S3 23.243 15.254 13.361 22.245 14.247 Table 9 Effect of different pH on production of L-methioninase during LSF S.No. Isolates Units of L-methioninase Production at different pH values maintained in LSF medium pH–4 pH–5 pH–6 pH–7 pH–8 1. S2 11.079 15.246 17.178 25.424 17.361 2. S3 12.188 15.119 16.346 21.758 18.079 Table 10 Effect of different on temperatures production of L-methioninase during LSF S.No. Isolates Units of L-methioninase Production at different temperature conditions maintained in LSF medium 20oC 25oC 30oC 35oC 40oC 1. S2 10.335 12.639 14.436 12.639 10.224 2. S3 11.424 15. 734 23.844 15.237 11.436 Table 11 In vitro L-methioninase production by selected Streptomyces spp. isolates at combined optimized fermentation parameters during LSF S.No. Streptomyces isolates Estimated L-Methioninase Produced in Units/ml 1. S2 30.76 2. S3 24.10 World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 266-275 272 Figure 2 Graphical description of L-methioninase enzyme production during liquid state fermentation activity by 2 selected indigenous Streptomyces spp. isolates due to influence of 5 different carbohydrates as a source of carbon Figure 3 Graphical description of L-methioninase enzyme production during liquid state fermentation activity by 2 selected indigenous Streptomyces spp. isolates under 5 different pH conditions World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 266-275 273 Figure 4 Graphical description of L-methioninase enzyme production during liquid state fermentation activity by 2 selected indigenous Streptomyces spp. isolates under 5 different thermal conditions in terms of degree centigrade Figure 5 Graphical description of L-Methioninase produced by 2 selected indigenous Streptomyces spp. isolates at combined optimum LSF parameters The present study focused on the production of L-methioninase by indigenous Streptomyces spp. isolates under liquid state fermentation (LSF) conditions, optimizing key factors such as carbon sources, pH, and temperature to enhance enzyme activity. L-methioninase has gained significant attention for its potential as an anticancer enzyme, and its efficient production is crucial for therapeutic applications (Xu et al., 2020b). The enumeration of target microbial species from various soil samples revealed considerable diversity in colonyforming units (CFU). Kamla Park, with 348 CFUs, demonstrated the highest microbial density, which may be attributed to the organic matter richness of the park soil, supporting microbial growth. This finding is consistent with studies indicating that nutrient-dense soils, such as those found in parks, promote the growth of Streptomyces spp., enhancing their enzymatic potential (Xu et al., 2020b). In contrast, other sites like Kaliasot Dam and Bairagarh showed lower microbial populations, suggesting environmental factors significantly influence microbial diversity and enzyme production potential. Morphological and biochemical characterizations of the isolates revealed typical Streptomyces features such as filamentous colonies and catalase-positive reactions. The variation in colony color, from creamy white to pinkish, further highlights the phenotypic diversity within the isolates, consistent with the findings of Kumar and Singh (2020). World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 266-275 274 The biochemical assays revealed that all isolates tested positive for citrate utilization and starch hydrolysis, indicating robust metabolic activity, although only two isolates, S1 and S4, tested positive for methyl red reduction, suggesting diversity in fermentation pathways (Bibb et al., 2018b). Regarding enzyme production, the LSF results indicated that carbon source, pH, and temperature play a crucial role in optimizing L-methioninase production. Sucrose was the most effective carbon source for enzyme production, yielding the highest enzyme activity for both isolates (S2: 21.236 U/mL; S3: 23.243 U/mL). Lactose also performed well, while mannitol and maltose were less effective, with maltose showing the lowest enzyme production. This highlights the importance of selecting appropriate carbon sources, as supported by Veeranagouda et al. (2015), who found that carbohydrate selection significantly influences enzyme yields in microbial systems. The optimal pH for L-methioninase production was determined to be pH 7, aligning with earlier research that indicates neutral pH enhances microbial enzymatic activity (Veeranagouda et al., 2015b). Acidic environments (pH 4 and pH 5) significantly inhibited enzyme production, likely due to their detrimental effects on microbial metabolism. For temperature, 30°C was identified as the optimal condition for enzyme production, particularly for isolate S3, which exhibited the highest enzyme activity (23.844 U/mL). Temperatures outside the mesophilic range led to reduced enzyme production, reflecting the temperature sensitivity of Streptomyces spp. enzyme systems. Overall, the optimized conditions for L-methioninase production in the present study—sucrose as the carbon source, pH 7, and 30°C—are consistent with prior studies on microbial enzyme optimization. These findings contribute valuable insights into the large-scale production potential of L-methioninase for therapeutic applications, particularly in anticancer treatments. 4. Conclusion This preliminary study successfully isolated and characterized indigenous Streptomyces spp. from the Upper Lake of Bhopal, demonstrating their potential for L-methioninase production under optimized liquid state fermentation conditions. The findings highlight sucrose, pH 7, and 30°C as optimal parameters for enhancing enzyme activity. These insights contribute to the growing bioprospecting efforts for microbial enzymes with therapeutic applications, particularly in cancer treatment, paving the way for future research and potential industrial applications of Lmethioninase. Compliance with ethical standards Disclosure of conflict of interest No conflict of interest to be disclosed. References [1] Bibb, M. J., Domonkos, A., Chandra, G., & Buttner, M. J. (2018a). Metabolic pathways of Streptomyces species and their biotechnological applications. Microbial Biotechnology, 11(4), 638-653. [2] Bibb, M.J., Hesketh, A., & Hong, H.J. (2018b). Streptomyces genetics and metabolism: Insights from functional genomics. Microbiology and Molecular Biology Reviews, 82(2), e00047-17. [3] El-Naggar, N. E., & Eldin, T. A. (2020). 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