Determination of Amylolytic and Proteolytic Enzyme Activity Characteristics of Thermophilic Actinobacteria Isolated from Amasya-Terziköy Thermal Spring
Year 2025,
Volume: 40 Issue: 4, 278 - 284, 10.10.2025
Demet Tatar
,
Aysel Veyisoğlu
,
Ali Tokatlı
,
Hünkar Avni Duyar
Abstract
Actinobacteria represent a group of Gram-positive microorganisms characterized by high G+C content and are commonly found in both terrestrial and aquatic ecosystems. These bacteria are notable for their capacity to synthesize a wide range of bioactive compounds, including antibiotics and industrial enzymes. Their ability to withstand extreme environmental conditions allows them to colonize challenging habitats, such as thermal springs. These geothermal environments are critical not only for harboring microbial diversity but also for the discovery of novel bioactive substances with therapeutic potential. This study focused on isolating thermophilic actinobacteria from Terziköy Thermal Spring in Amasya, Türkiye, followed by phylogenetic analysis through 16S rRNA gene sequencing. The enzymatic capacities of the isolates—specifically amylase and protease production—were also assessed. Fourteen of the 21 isolates underwent 16S rRNA sequencing, resulting in the identification of eight Streptomyces, four Nocardia, one Actinomadura, and one Pseudonocardia species. Among these, seven isolates demonstrated amylase activity, and one displayed protease production. Notably, one Streptomyces isolate exhibited both enzymatic activities. These results highlight the phylogenetic diversity of thermophilic actinobacteria in geothermal environments and their potential as sources of thermostable enzymes for industrial applications.
Ethical Statement
Ethics committee approval was not required for this study. All authors declare that no experiments involving human or animal subjects were conducted.
Supporting Institution
Ethics committee approval was not required for this study. All authors declare that no experiments involving human or animal subjects were conducted.
Project Number
This study was supported by Hitit University Scientific Research Projects Coordination Department with project number ODMYO19001.23.002.
Thanks
The authors would like to express their sincere gratitude to the Hitit University Scientific Research Projects Coordination Department for providing financial support.
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Ertaş, H. B., Kiliç, A., Özbey, G., & Muz, A. (2005). Isolation of Arcanobacterium (Actinomyces) pyogenes from abscessed cattle kidney and identification by PCR. Turk J Vet Anim Sci, 29(2), 455–459. https://journals.tubitak.gov.tr/ veterinary/vol29/iss2/39 google scholar
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Felsenstein, J. (1985). Confidence limits on phylogeny: An appropriate use of the bootstrap. Evolution, 39, 783–791. https://doi.org/10.2307/2408678 google scholar
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Fossi, B.T., Tavea, F., Jiwoua, C., & Ndjouenke, R. (2009). Screening and phenotypic characterization of thermostable amylases producing yeasts and bacteria strains from some Cameroonian soils. African Journal of Microbiology Research, 3(9), 504–514. google scholar
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Goodfellow, M. & Williams, S. T. (1983). Ecology of Actinomycetes. Annu Rev Microbiol, 37, 189–216. https://doi.org/10.1146/annurev.mi.37.100183.001201 google scholar
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Gorrab, A., Ouertani, R., Souii, A., Kallel, F., Masmoudi, A. S., Cherif, A., & Neifar, M. (2024). A comprehensive review on the properties and applications of extremozymes from extremophilic actinobacteria. MOJ Applied Bionics and Biomechanics, 8(1), 76-85. https://doi.org/10.15406/mojabb.2024.08.00213 google scholar
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Jones, K. L. (1949). Fresh isolates of Actinomycetes in which the presence of sporogenous aerial mycelia is a fluctuating characteristic. Journal of Bacteriology, 57, 141–145. https://doi.org/10.1128/jb.57.2.141-145 google scholar
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Könönen, E., & Wade, W. G. (2015). Actinomyces and related organisms in human infections. Clin Microbiol Rev, 28(2):419–442. https://doi.org/10.1128/CMR. 00100-14 google scholar
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Kumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S., & Tamura, K. (2024). Molecular evolutionary genetics analysis version 12 for adaptive and green computing. Molecular Biology and Evolution, 41(12), 1–9. https://doi.org/10. 1093/molbev/msae263 google scholar
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Lerat, S., Simao-Beaunoir, A. M., & Beaulieu, C. (2009). Genetic and physiological determinants of Streptomyces scabies pathogenicity. Molecular Plant Pathology. 1 0, 579–585. https://doi.org/10.1111/j.1364-3703.2009.00561.x google scholar
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Ma, G., Xia, Z., & Wu, S. (2009). Inhibiting effect of seven marine actinomycete strains against vegetable pathogenic microorganisms. Crops, 5, 3–9. google scholar
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Medjemadj, M., Escuder-Rodríguez, J. J., Boudemagh, A., & González-Siso, M. I. (2020). Actinobacteria isolated from Algerian hot spring waters: A potential source of important enzymes. Eco. Env. & Cons, 26(3), 1145-1157. google scholar
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Mohamedin, A. H. (1999). Isolation, identification and some cultural conditions of a protease-producing thermophilic Streptomyces strain grown on chicken feather as a substrate. International Biodeterioration and Biodegradation, 43, 13–21. https://doi.org/10.1016/S0964-8305(98)00061-4 google scholar
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Oztas Gulmus, E., & Gormez, A. (2020). Identification and characterization of novel thermophilic bacteria from hot springs, Erzurum, Turkey. Curr Microbiol, 77(6), 979–987. https://doi.org/10.1007/s00284-020-01880-0 google scholar
-
Pathom-Aree, W., Stach, J. E., Ward, A. C., Horikoshi, K., Bull, A. T., & Goodfellow, M. (2006). Diversity of actinomycetes isolated from Challenger Deep sediment (10,898 m) from the Mariana Trench. Extremophiles, 10(3), 181–189. https://doi. org/10.1007/s00792-005-0482-z google scholar
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Rego, A., Raio, F., Martins, T. P., Ribeiro, H., Sousa, A. G. G., Séneca, J., Baptista, M. S., Lee, C. K., Cary, S. C., Ramos, V., Carvalho, M. F., Leão, P. N., & Magalhães, C. (2019). Actinobacteria and Cyanobacteria diversity in terrestrial Antarctic microenvironments evaluated by culture-dependent and independent methods. Front Microbiol, 10, 1018. https://doi.org/10.3389/fmicb. 2019.01018 google scholar
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Saitou, N., & Nei, M. (1987). The neighbour-joining method: A new method for constructing phylogenetic trees. Molecular Biology and Evolution, 4(4), 406– 425. https://doi.org/10.1093/oxfordjournals.molbev.a040454 google scholar
-
Shirling, E. B., & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. International Journal of Systematic Bacteriology, 16, 313–340. https:// doi.org/10.1099/00207713-16-3-313 google scholar
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Shivlata, L., & Satyanarayana, T. (2015). Thermophilic and alkaliphilic Actinobacteria: Biology and potential applications. Frontiers in Microbiology, 6, 1014. https:// doi.org/10.3389/fmicb.2015.01014 google scholar
-
Shomali, B. A., & Danish-Daniel, M. (2024). Review on bioprospecting of thermophilic enzymes from hot springs via omics approaches. AACL Bioflux, 17(5), 2156-2174. Retrieved from https://bioflux.com.ro/docs/2024.2156-2174.pdf google scholar
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Stackebrandt, E., & Ebers, J. (2006). Taxonomic parameters revisited: tarnished gold standards. Microbiol Today, 33, 152–155. google scholar
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Stackebrandt, E., & Goebel, B. (1994). Taxonomic note: A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. International Journal of Systematic Bacteriology, 44(4), 846–849. https://doi.org/10.1099/00207713-44-4-846 google scholar
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Takahashi, Y., & Omura, S. (2003). Isolation of new actinomycete strains for the screening of new bioactive compounds. J Gen Appl Microbiol, 49(3), 141–154. https://doi.org/10.2323/jgam.49.141 google scholar
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Tang, H., Kambris, Z., Lemaitre, B., & Hashimoto, C. (2006). Two proteases defining a melanization cascade in the immune system of Drosophila. J Biol Chem, 281(38), 28097-28104. https://doi.org/10.1074/jbc.M601642200 google scholar
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Veyisoglu, A., & Tatar, D. (2021). Diversity and antimicrobial activity of culturable actinobacteria isolated from the sediment of Sarıkum Lake. Biotechnology & Biotechnological Equipment, 35(1), 1136–1146. https://doi.org/10.1080/ 13102818.2021.1952898 google scholar
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Veyisoglu, A., Tatar, D., Duyar, H. A., & Tokatli, A. (2024). Isolation, molecular characterization and determination of antagonistic properties of alkalitolerant Streptomyces members from Van lake-Çarpanak island soil. Marine Science and Technology Bulletin, 13(3), 183–198. https://doi.org/10. 33714/masteb.1522501 google scholar
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Yoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, H., & Chun, J. (2017). Introducing EzBioCloud: A taxonomically united daltabansa of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol, 67(5), 1613–1617. https://doi.org/10.1099/ ijsem.0.001755 google scholar
Year 2025,
Volume: 40 Issue: 4, 278 - 284, 10.10.2025
Demet Tatar
,
Aysel Veyisoğlu
,
Ali Tokatlı
,
Hünkar Avni Duyar
Project Number
This study was supported by Hitit University Scientific Research Projects Coordination Department with project number ODMYO19001.23.002.
References
-
Aksoy, S. Ç., Uzel, A. & Hameş Kocabaş, E. E. (2012). Extracellular serine proteases produced by Thermoactinomyces strains from hot springs and soils of West Anatolia. Ann Microbiol, 62, 483–492. https://doi.org/10.1007/s13213-011-0280-z google scholar
-
Amasya İl Kültür ve Turizm Müdürlüğü (2025, July 31), https://amasya.ktb.gov.tr/TR-243587/terzikoy-termal.html google scholar
-
Bérdy, J. (2005). Bioactive microbial metabolites. J Antibiot, 58(4), 1–26. https://doi. org/10.1038/ja.2005.1 google scholar
-
Buchholz-Cleven, B. E. E., Rattunde, B., & Straub, K. L. (1997). Screening for genetic diversity of isolates of anaerobic Fe(II)-oxidizing bacteria using DGGE and whole-cell hybridization. Systematic and Applied Microbiology, 20, 301–309. https://doi.org/10.1016/S0723-2020(97)80077-X google scholar
-
Busarakam, K., Bull, A. T., Trujillo, M. E., Riesco, R., Sangal, V., van Wezel, G. P., & Goodfellow, M. (2016). Modestobacter caceresii sp. nov., novel actinobacteria with an insight into their adaptive mechanisms for survival in extreme hyperarid Atacama Desert soils. Syst Appl Microbiol, 39(4), 243–251. https://doi.org/ 10.1016/j.syapm.2016.03.007 google scholar
-
Challis, G. L., & Hopwood, D. A. (2003). Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc Natl Acad Sci U S A100, (Suppl 2), 14555–14561. https://doi.org/10. 1073/pnas.1934677100 google scholar
-
Chun, J. (1995). Computer Assisted Classification and Identification of Actinomycetes. PhD thesis. Department of Microbiology, University of Newcastle, Newcastle upon Tyne, UK. google scholar
-
Elbendary, A. A., Hessain, A. M., El-Hariri, M. D., Seida, A. A., Moussa, I. M., Mubarak, A. S., Kabli, S. A., Hemeg, H.A. & El Jakee, J.K. (2018). Isolation of antimicrobial producing Actinobacteria from soil samples. Saudi J Biol Sci, 25(1), 44–46. https://doi.org/10.1016/j.sjbs.2017.05.003 google scholar
-
Ertaş, H. B., Kiliç, A., Özbey, G., & Muz, A. (2005). Isolation of Arcanobacterium (Actinomyces) pyogenes from abscessed cattle kidney and identification by PCR. Turk J Vet Anim Sci, 29(2), 455–459. https://journals.tubitak.gov.tr/ veterinary/vol29/iss2/39 google scholar
-
Felsenstein, J. (1985). Confidence limits on phylogeny: An appropriate use of the bootstrap. Evolution, 39, 783–791. https://doi.org/10.2307/2408678 google scholar
-
Fossi, B.T., Tavea, F., Jiwoua, C., & Ndjouenke, R. (2009). Screening and phenotypic characterization of thermostable amylases producing yeasts and bacteria strains from some Cameroonian soils. African Journal of Microbiology Research, 3(9), 504–514. google scholar
-
Goodfellow, M. & Williams, S. T. (1983). Ecology of Actinomycetes. Annu Rev Microbiol, 37, 189–216. https://doi.org/10.1146/annurev.mi.37.100183.001201 google scholar
-
Gorrab, A., Ouertani, R., Souii, A., Kallel, F., Masmoudi, A. S., Cherif, A., & Neifar, M. (2024). A comprehensive review on the properties and applications of extremozymes from extremophilic actinobacteria. MOJ Applied Bionics and Biomechanics, 8(1), 76-85. https://doi.org/10.15406/mojabb.2024.08.00213 google scholar
-
Jones, K. L. (1949). Fresh isolates of Actinomycetes in which the presence of sporogenous aerial mycelia is a fluctuating characteristic. Journal of Bacteriology, 57, 141–145. https://doi.org/10.1128/jb.57.2.141-145 google scholar
-
Könönen, E., & Wade, W. G. (2015). Actinomyces and related organisms in human infections. Clin Microbiol Rev, 28(2):419–442. https://doi.org/10.1128/CMR. 00100-14 google scholar
-
Kumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S., & Tamura, K. (2024). Molecular evolutionary genetics analysis version 12 for adaptive and green computing. Molecular Biology and Evolution, 41(12), 1–9. https://doi.org/10. 1093/molbev/msae263 google scholar
-
Küster, E., & Williams, S. T. (1964). Selection of media for isolation of streptomycetes. Nature, 202, 928–929. https://doi.org/10.1038/202928a0 google scholar
-
Lacey, J. (1988). Actinomycetes as biodeteriogenes pollutants of the environment. In: Actinomycetes in Biotechnology, Goodfellow, M., Williams, S., Mordarski, M., Eds. Academic Press. San Diego: 359–417. google scholar
-
Lerat, S., Simao-Beaunoir, A. M., & Beaulieu, C. (2009). Genetic and physiological determinants of Streptomyces scabies pathogenicity. Molecular Plant Pathology. 1 0, 579–585. https://doi.org/10.1111/j.1364-3703.2009.00561.x google scholar
-
Ma, G., Xia, Z., & Wu, S. (2009). Inhibiting effect of seven marine actinomycete strains against vegetable pathogenic microorganisms. Crops, 5, 3–9. google scholar
-
Medjemadj, M., Escuder-Rodríguez, J. J., Boudemagh, A., & González-Siso, M. I. (2020). Actinobacteria isolated from Algerian hot spring waters: A potential source of important enzymes. Eco. Env. & Cons, 26(3), 1145-1157. google scholar
-
Mohamedin, A. H. (1999). Isolation, identification and some cultural conditions of a protease-producing thermophilic Streptomyces strain grown on chicken feather as a substrate. International Biodeterioration and Biodegradation, 43, 13–21. https://doi.org/10.1016/S0964-8305(98)00061-4 google scholar
-
Oztas Gulmus, E., & Gormez, A. (2020). Identification and characterization of novel thermophilic bacteria from hot springs, Erzurum, Turkey. Curr Microbiol, 77(6), 979–987. https://doi.org/10.1007/s00284-020-01880-0 google scholar
-
Pathom-Aree, W., Stach, J. E., Ward, A. C., Horikoshi, K., Bull, A. T., & Goodfellow, M. (2006). Diversity of actinomycetes isolated from Challenger Deep sediment (10,898 m) from the Mariana Trench. Extremophiles, 10(3), 181–189. https://doi. org/10.1007/s00792-005-0482-z google scholar
-
Rego, A., Raio, F., Martins, T. P., Ribeiro, H., Sousa, A. G. G., Séneca, J., Baptista, M. S., Lee, C. K., Cary, S. C., Ramos, V., Carvalho, M. F., Leão, P. N., & Magalhães, C. (2019). Actinobacteria and Cyanobacteria diversity in terrestrial Antarctic microenvironments evaluated by culture-dependent and independent methods. Front Microbiol, 10, 1018. https://doi.org/10.3389/fmicb. 2019.01018 google scholar
-
Saitou, N., & Nei, M. (1987). The neighbour-joining method: A new method for constructing phylogenetic trees. Molecular Biology and Evolution, 4(4), 406– 425. https://doi.org/10.1093/oxfordjournals.molbev.a040454 google scholar
-
Shirling, E. B., & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. International Journal of Systematic Bacteriology, 16, 313–340. https:// doi.org/10.1099/00207713-16-3-313 google scholar
-
Shivlata, L., & Satyanarayana, T. (2015). Thermophilic and alkaliphilic Actinobacteria: Biology and potential applications. Frontiers in Microbiology, 6, 1014. https:// doi.org/10.3389/fmicb.2015.01014 google scholar
-
Shomali, B. A., & Danish-Daniel, M. (2024). Review on bioprospecting of thermophilic enzymes from hot springs via omics approaches. AACL Bioflux, 17(5), 2156-2174. Retrieved from https://bioflux.com.ro/docs/2024.2156-2174.pdf google scholar
-
Stackebrandt, E., & Ebers, J. (2006). Taxonomic parameters revisited: tarnished gold standards. Microbiol Today, 33, 152–155. google scholar
-
Stackebrandt, E., & Goebel, B. (1994). Taxonomic note: A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. International Journal of Systematic Bacteriology, 44(4), 846–849. https://doi.org/10.1099/00207713-44-4-846 google scholar
-
Takahashi, Y., & Omura, S. (2003). Isolation of new actinomycete strains for the screening of new bioactive compounds. J Gen Appl Microbiol, 49(3), 141–154. https://doi.org/10.2323/jgam.49.141 google scholar
-
Tang, H., Kambris, Z., Lemaitre, B., & Hashimoto, C. (2006). Two proteases defining a melanization cascade in the immune system of Drosophila. J Biol Chem, 281(38), 28097-28104. https://doi.org/10.1074/jbc.M601642200 google scholar
-
Veyisoglu, A., & Tatar, D. (2021). Diversity and antimicrobial activity of culturable actinobacteria isolated from the sediment of Sarıkum Lake. Biotechnology & Biotechnological Equipment, 35(1), 1136–1146. https://doi.org/10.1080/ 13102818.2021.1952898 google scholar
-
Veyisoglu, A., Tatar, D., Duyar, H. A., & Tokatli, A. (2024). Isolation, molecular characterization and determination of antagonistic properties of alkalitolerant Streptomyces members from Van lake-Çarpanak island soil. Marine Science and Technology Bulletin, 13(3), 183–198. https://doi.org/10. 33714/masteb.1522501 google scholar
-
Yoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, H., & Chun, J. (2017). Introducing EzBioCloud: A taxonomically united daltabansa of 16S rRNA and whole genome assemblies. Int J Syst Evol Microbiol, 67(5), 1613–1617. https://doi.org/10.1099/ ijsem.0.001755 google scholar