Molecular identification of cellulase and protease producing Bacillus tequilensis UTMSA14 isolated from the geothermal hot spring in Lau Sidebuk Debuk, North Sumatra, Indonesia
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Abstract. Fachrial E, Putri RRJS, Lister INE, Anggraini S, Harmileni, Nugroho TT, Saryono. 2020. Molecular identification of cellulase and protease producing Bacillus tequilensis UTMSA14 isolated from the geothermal hot spring in Lau Sidebuk Debuk, North Sumatra, Indonesia. Biodiversitas 21: 4719-4725. The industrial need for stable microbial enzymes tends to increase every year. The aim of this study was to isolate and identify the protease and cellulase producing thermophilic bacteria isolated from the geothermal spring of Lau Sidebuk Debuk, North Sumatra, Indonesia. The protease activity was determined based on the formation of halo zone on Skim Milk Agar, while cellulase was confirmed using CMC plate agar method. Nine bacterial isolates were successfully obtained from the sediments and water, and both activities were observed in only one isolate, known as UTMSA14. These isolates were characterized biochemically and morphologically. The molecular identification of UTMSA14 was done by 16S rRNA sequencing based on BLAST and phylogenetic analysis using MEGA X. Phylogenetic analysis showed that UTMSA14 clustered together with Bacillus tequilensis strain 10b (accession number NR_104919.1), which ascertained the isolate as Bacillus tequilensis strain 10b (accession number NR_104919.1). This is the first report of a thermophilic bacteria isolated from Lau Sidebuk Debuk hot springs, with the ability to produce cellulase and protease. further research is needed to purify and characterize the resulting enzymes, with a potential for industrial applications.
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References
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Acharya, S., & Chaudhary, A. (2012). Bioprospecting thermophiles for cellulase production: A review. Brazilian Journal of Microbiology, 43(3), 844–856. https://doi.org/10.1590/S1517-83822012000300001
Adhikari, H., Ghimire, S., Khatri, B., & K.C., Y. (2015). Enzymatic Screening and Molecular Characterization of Thermophilic Bacterial Strains Isolated from Hotspring of Tatopani, Bhurung, Nepal. International Journal of Applied Sciences and Biotechnology, 3(3), 392–397. https://doi.org/10.3126/ijasbt.v3i3.12724
Aditiawati, P., Yohandini, H., Madayanti, F., & Akhmaloka. (2009). Microbial Diversity of Acidic Hot Spring (Kawah Hujan B) in Geothermal Field of Kamojang Area, West Java-Indonesia. The Open Microbiology Journal, 3(1), 58–66. https://doi.org/10.2174/1874285800903010058
Agustina, W., Rahman, T., Ratnawati, L., Sriharti, Salim, T., & Nursyahbani, I. (2019). Potential Isolates Characterization of Thermophilic Bacteria from Hot Springs and Waste Agricultural Production in Subang District Area. IOP Conference Series: Earth and Environmental Science, 251(1), 1–7. https://doi.org/10.1088/1755-1315/251/1/012006
Alam, M. G., Uddin, M. E., Rahman, S., Karim, M. R., Islam, M. S., Ahamad, T., Hossain, M. J., Mahmood, M. S., Alam, M. S., Maitra, P., & Nazmuzzaman, M. (2017). Protease activity of extracellular enzyme produced by B. subtilis isolated from soil. International Journal of Environment, Agriculture and Biotechnology, 2(1), 382–388. https://doi.org/10.22161/ijeab/2.1.48
Br. Sinulingga, E. K., Humaizi, H., & Lubis, Z. (2019). The Role of Community Participation in the Development of Lau Debuk-debuk Hot Spring in Doulu village, Karo regency. International Journal of Multicultural and Multireligious Understanding, 6(5), 16. https://doi.org/10.18415/ijmmu.v6i5.1021
Fachrial, E., Anggraini, S., Harmileni, Nugroho, T. T., & Saryono. (2019). Isolation and molecular identification of carbohydrase and protease producing Bacillus subtilis JCM 1465 isolated from Penen Hot Springs in North Sumatra, Indonesia. Biodiversitas, 20(12), 3493–3498. https://doi.org/10.13057/biodiv/d201205
Fukui, Y., & Sawabe, T. (2007). Improved One-step Colony PCR Detection of Vibrio harveyi. Microbes and Environments, 22(1), 1–10. https://doi.org/10.1264/jsme2.22.1
Ifandi, S., & Alwi, M. (2018). Isolation of Thermophilic Bacteria from Bora Hot Springs in Central Sulawesi. Biosaintifika: Journal of Biology & Biology Education, 10(2), 291–297. https://doi.org/10.15294/biosaintifika.v10i2.14905
Jannah, A., Aulanniam, Ardyati, T., & Suharjono. (2018). Isolation, cellulase activity test and molecular identification of selected cellulolytic bacteria indigenous rice bran. Indonesian Journal of Chemistry, 18(3), 514–521. https://doi.org/10.22146/ijc.26783
Khan, Z., Shafique, M., Nawaz, H. R., Jabeen, N., & Naz, S. A. (2019). Bacillus tequilensis ZMS-2: A novel source of alkaline protease with antimicrobial, anti-coagulant, fibrinolytic and dehairing potentials. Pakistan Journal of Pharmaceutical Sciences, 32(4), 1913–1918.
Li, H., Guan, Y., Dong, Y., Zhao, L., Rong, S., Chen, W., Lv, M., Xu, H., Gao, X., Chen, R., Li, L., & Xu, Z. (2018). Isolation and evaluation of endophytic Bacillus tequilensis GYLH001 with potential application for biological control of Magnaporthe oryzae. PLoS ONE, 13(10), 1–18. https://doi.org/10.1371/journal.pone.0203505
Mehta, R., Singhal, P., Singh, H., Damle, D., & Sharma, A. K. (2016). Insight into thermophiles and their wide-spectrum applications. 3 Biotech, 6(1), 1–9. https://doi.org/10.1007/s13205-016-0368-z
Missa, H., Susilowati, A., & Setyaningsih, R. (2016). Diversity and phylogenetic relationship of cellulolytic bacteria from the feces of Bali Cattle in South Central Timor, East Nusa Tenggara, Indonesia. Biodiversitas, 17(2), 614–619. https://doi.org/10.13057/biodiv/d170232
Mohammad, B. T., Al Daghistani, H. I., Jaouani, A., Abdel-Latif, S., & Kennes, C. (2017). Isolation and Characterization of Thermophilic Bacteria from Jordanian Hot Springs: Bacillus licheniformis and Thermomonas hydrothermalis Isolates as Potential Producers of Thermostable Enzymes. International Journal of Microbiology, 2017, 1–12. https://doi.org/10.1155/2017/6943952
Panda, M. K., Sahu, M. K., & Tayung, K. (2013). Isolation and characterization of a thermophilic Bacillus sp. with protease activity isolated from hot spring of Tarabalo, Odisha, India. Iranian Journal of Microbiology, 5(2), 159–165.
Razzaq, A., Shamsi, S., Ali, A., Ali, Q., Sajjad, M., Malik, A., & Ashraf, M. (2019). Microbial proteases applications. Frontiers in Bioengineering and Biotechnology, 7(JUN), 1–20. https://doi.org/10.3389/fbioe.2019.00110
Saggu, S. K., & Mishra, P. C. (2017). Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil. PLoS ONE, 12(11), 1–18. https://doi.org/10.1371/journal.pone.0188724
Simon, N., Unjah, T., Yusry, M., & Dzulkafli, M. A. (2019). Physico-chemical characterisation and potential health benefit of the hulu langat hot spring in selangor, Malaysia. Sains Malaysiana, 48(11), 2451–2462. https://doi.org/10.17576/jsm-2019-4811-15
Srinivasan, R., Karaoz, U., Volegova, M., MacKichan, J., Kato-Maeda, M., Miller, S., Nadarajan, R., Brodie, E. L., & Lynch, S. V. (2015). Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens. PLoS ONE, 10(2), 1–22. https://doi.org/10.1371/journal.pone.0117617
Yd, J., Vinothkumar, R., Ab, I., Nm, I., Ganie, P. A., Hussain, R. A. B., & Vennila, A. (2019). Isolation and characterization of thermophilic bacteria from Maharashtra hot springs: Bacillus sp. and Staphylococcus sp. Journal of Entomology and Zoology Studies, 7(1), 691–695.
Yohandini, H., Julinar, & Muharni. (2015). Isolation and Phylogenetic Analysis of Thermophile Community Within Tanjung Sakti Hot Spring, South Sumatera, Indonesia. HAYATI Journal of Biosciences, 22(3), 143–148. https://doi.org/10.1016/j.hjb.2015.10.006