BELEDİYE ANAEROBİK ARITMA SİSTEMİNDEN İZOLE EDİLEN YENİ BİR ANAEROBİK BAKTERİ İLE ALKALİ PROTEAZ ÜRETİMİ

Öz

Bu çalışmada, anaerobik arıtma sisteminden toplanan çamur örneklerinden izole edilen bir bakteri suşunun alkali proteaz enzim üretimi araştırılmıştır. 16S rDNA dizi analizine göre, izolatın Thermoanaerobacter thermohidrosulfuricus (%98.52) olduğu tespit edilmiştir. Enzim aktivitesi analizleri, optimum pH değerinin 10, inkübasyon süresinin 64 saat ve sıcaklığın 35°C olduğunu ortaya çıkarmıştır. Arabinoz ve kazein hidrolizatlarının sırasıyla en iyi karbon ve nitrojen kaynakları olduğu bulunmuştur. Maksimum proteaz aktivitesi (864.68 U/mL) glikoz yerine arabinoz kullanıldığında kaydedilmiştir. Ayrıca, besiyerine 1 g/L MgSO4.7H2O ve 0.25 g/L Tween-80 ilavesi enzim aktivitesini artırmıştır. Bu nedenle, T. thermohidrosulfuricus’un kültür ortamında önemli bir alkali proteaz enzimi üreticisi olduğu sonucuna varılabilmektedir. Bildiğimiz kadarıyla bu çalışma, T. thermohidrosulfuricus’un alkali proteaz üretiminin optimizasyonunu araştıran ilk çalışmadır.

Anahtar Kelimeler

• Hücre dışı
• proteolitik aktivite
• enzim aktivitesi
• yeni izolat
• Thermoanaerobacter

PRODUCTION OF ALKALINE PROTEASE BY A NOVEL ANAEROBIC BACTERIUM ISOLATED FROM A MUNICIPAL ANAEROBIC TREATMENT SYSTEM

Öz

In this study, alkaline protease enzyme production by a bacterial strain isolated from sludge samples collected from an anaerobic treatment system was investigated. According to the 16S rDNA sequence analysis, the isolate was identified as Thermoanaerobacter thermohydrosulfuricus (98.52%). Enzyme activity analyses revealed an optimum pH value of 10, an incubation time of 64 h, and a temperature of 35°C. Arabinose and casein hydrolysates were found to be the best carbon and nitrogen sources, respectively. Maximum protease activity was recorded (864.68 U/mL) when arabinose was used instead of glucose. Moreover, the addition of 1 g/L MgSO4.7H2O and 0.25 g/L Tween-80 to the medium increased the enzyme activity. Therefore, it can be concluded that T. thermohydrosulfuricus is a significant producer of alkaline protease enzymes in the culture medium. To the best of our knowledge, this is the first study to investigate the optimization of alkaline protease production by T. thermohydrosulfuricus.

Anahtar Kelimeler

• Extracellular
• proteolytic activity
• enzyme activity
• new isolate
• Thermoanaerobacter

Kaynakça

1. Aftab, S., Ahmed, S., Saeed, S., Rasool, S. A. (2006). Screening, isolation and characterization of alkaline protease producing bacteria from soil. Pakistan Journal of Biological Sciences, 9(11), 2122-2126.
2. Akcan, N., Uyar, F. (2011). Production of extracellular alkaline protease from Bacillus subtilis RSKK96 with solid state fermentation. EurAsian Journal of BioSciences, 5,64-71. https://doi.org/ 10.5053/ejobios.2011.5.0.8
3. Akhter, K., Kiani, H. A., Ghous, T., Rasheed, A., Gillani, H., Akhtar, T. (2024). Production, partial purification and optimization of oilseed-based protease and its application as an efficient eco-friendly alternative for destaining and dehairing process. Waste and Biomass Valorization, 1-10. https://doi.org/10.1007/s12649-024-02438-y
4. Al-Dhabi, N. A., Esmail, G. A., Ghilan, A. K. M., Arasu, M. V., Duraipandiyan, V., Ponmurugan, K. (2020). Characterization and fermentation optimization of novel thermo stable alkaline protease from Streptomyces sp. Al-Dhabi-82 from the Saudi Arabian environment for eco-friendly and industrial applications. Journal of King Saud University-Science, 32(1),1258-1264. https://doi.org/10.1016/j.jksus.2019.11.011
5. Aqel, H., Al-Quadan, F., Yousef, T. K. (2012). A novel neutral protease from thermophilic Bacillus strain HUTBS62. Journal of Bioscience & Biotechnology, 1(2).
6. Arya, P. S., Yagnik, S. M., Rajput, K. N., Panchal, R. R., Raval, V. H. (2021). Understanding the basis of occurrence, biosynthesis, and implications of thermostable alkaline proteases. Applied Biochemistry and Biotechnology, 1-38. https://doi.org/10.1007/s12010-021-03701-x
7. Asha, B., Palaniswamy, M. (2018). Optimization of alkaline protease production by Bacillus cereus FT 1isolated from soil. Journal of Applied Pharmaceutical Science, 8(2), 119-127. https://doi.org/10.7324/JAPS.2018.8219
8. Banerjee, U. C., Sani, R. K., Azmi, W., Soni, R. (1999). Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process Biochemistry, 35(1-2), 213-219. https://doi.org/10.1016/S0032-9592(99)00053-9

9. Bashir, F., Asgher, M., Hussain, F., Randhawa, M. A. (2018). Development and characterization of cross-linked enzyme aggregates of thermotolerant alkaline protease from Bacillus licheniformis. International Journal of Biological Macromolecules, 113, 944-951. https://doi.org/ 10.1016/j.ijbiomac.2018.03.009
10. Charles, P., Devanathan, V., Anbu, P., Ponnuswamy, M. N., Kalaichelvan, P. T., Hur, B. K. (2008). Purification, characterization and crystallization of an extracellular alkaline protease from Aspergillus nidulans HA‐10. Journal of Basic Microbiology, 48(5), 347-352. https://doi.org/ 10.1002/jobm.200800043
11. Chauhan, R. S., Mishra, R. M. (2020). Characterization of alkaline protease producing Bacillus halodurans RSCVS-PF21 isolated from poultry farm soil. Biosciences Biotechnology Research Asia, 17(2), 385-392. http://dx.doi.org/ 10.13005/bbra/2841
12. Chi, Z., Ma, C., Wang, P., Li, H. F. (2007). Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans. Bioresource Technology, 98(3), 534-538. https://doi.org/ 10.1016/j.biortech.2006.02.006
13. Cupp-Enyard, C. (2008). Sigma’s non-specific protease activity assay-casein as a substrate. JoVE (Journal of Visualized Experiments), (19), e899. https://doi.org/10.3791/899
14. Datta, S., Menon, G., Varughese, B. (2017). Production, characterization, and immobilization of partially purified surfactant–detergent and alkali-thermostable protease from newly isolated Aeromonas caviae. Preparative Biochemistry and Biotechnology, 47(4), 349-356. https://doi.org/ 10.1080/10826068.2016.1244688
15. Denizci, A. A., Kazan, D., Abeln, E. C. A., Erarslan, A. (2004). Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under higly alkaline conditions. Journal of Applied Microbiology, 96(2), 320-327. https://doi.org/10.1046/j.1365-2672.2003.02153.x
16. Dhandapani, R., Vijayaragavan, R. (1994). Production of a thermophilic, extracellular alkaline protease by Bacillus stearothermophilus AP-4. World Journal of Microbiology and Biotechnology, 10, 33-35. https://doi.org/10.1007/BF00357559
17. Dorra, G., Ines, K., Imen, B. S., Laurent, C., Sana, A., Olfa, T., Pascal, C., Thierry, J., Ferid, L. (2018). Purification and characterization of a novel high molecular weight alkaline protease produced by an endophytic Bacillus halotolerans strain CT2. International Journal of Biological Macromolecules, 111, 342-351. https://doi.org/10.1016/ j.ijbiomac.2018.01.024
18. Elbanna, K., Ibrahim, I. M., Revol-Junelles, A. M. (2015). Purification and characterization of halo-alkali-thermophilic protease from Halobacterium sp. strain HP25 isolated from raw salt, Lake Qarun, Fayoum, Egypt. Extremophiles, 19, 763-774. https://doi.org/10.1007/s00792-015-0752-3
19. Esakkiraj, P., Immanuel, G., Sowmya, S. M., Iyapparaj, P., Palavesam, A. (2009). Evaluation of protease-producing ability of fish gut isolate Bacillus cereus for aqua feed. Food and Bioprocess Technology, 2, 383-390. https://doi.org/10.1007/ s11947-007-0046-6
20. Farooq, S., Nazir, R., Ganai, S. A., Ganai, B. A. (2021). Isolation and characterization of a new cold-active protease from psychrotrophic bacteria of Western Himalayan glacial soil. Scientific Reports, 11(1), 12768. https://doi.org/10.1038/ s41598-021-92197-w
21. Fujiwara, N., Masui, A., Imanaka, T. (1993). Purification and properties of the highly thermostable alkaline protease from an alkaliphilic and thermophilic Bacillus sp. Journal of Biotechnology, 30(2), 245-256. https://doi.org/ 10.1016/0168-1656(93)90117-6
22. Gençkal, H., Tari, C. (2006). Alkaline protease production from alkalophilic Bacillus sp. isolated from natural habitats. Enzyme and Microbial Technology, 39(4), 703-710. https://doi.org/ 10.1016/j.enzmictec.2005.12.004
23. Hammami, A., Hamdi, M., Abdelhedi, O., Jridi, M., Nasri, M., Bayoudh, A. (2017). Surfactant-and oxidant-stable alkaline proteases from Bacillus invictae: characterization and potential applications in chitin extraction and as a detergent additive. International Journal of Biological Macromolecules, 96, 272-281. https://doi.org/ 10.1016/j.ijbiomac.2016.12.035
24. Hashmi, S., Iqbal, S., Ahmed, I., Janjua, H. A. (2022). Production, optimization, and partial purification of alkali-thermotolerant proteases from newly isolated Bacillus subtilis S1 and Bacillus amyloliquefaciens KSM12. Processes, 10(6), 1050. https://doi.org/10.3390/pr10061050
25. Horikoshi, K. (1996). Alkaliphiles-from an industrial point of view. FEMS Microbiology Reviews, 18(2-3), 259-270. https://doi.org/ 10.1111/j.1574-6976.1996.tb00242.x
26. Hutadilok-Towatana, N., Painupong, A., Suntinanalert, P. (1999). Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS719. Journal of Bioscience and Bioengineering, 87(5), 581-587. https://doi.org/10.1016/s1389-1723(99)80118-2
27. Ibrahim, A. S., EI-Shayeb, N. M., Mabrouk, S. S. (2007). Isolation and identification of alkaline protease producing alkaliphilic bacteria from an Egyptian Soda Lake. Journal of Applied Sciences Research, 3(11), 1363-1368.
28. Jadhav, H. P., Sonawane, M. S., Khairnar, M. H., Sayyed, R. Z. (2020). Production of alkaline protease by rhizospheric Bacillus cereus HP_RZ17 and Paenibacillus xylanilyticus HP_RZ19. Environmental Sustainability, 3(1), 5-13. https://doi.org/10.1007/s42398-020-00096-z
29. Johnvesly, B., Naik, G. R. (2001). Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99 in a chemically defined medium. Process Biochemistry, 37(2), 139-144. http://doi.org/ 10.1016/S0032-9592(01)00191-1
30. Joo, H. S., Kumar, C. G., Park, G. C., Kim, K. T., Paik, S. R., Chang, C. S. (2002). Optimization of the production of an extracellular alkaline protease from Bacillus horikoshii. Process Biochemistry, 38(2), 155-159. http://doi.org/ 10.1016/S0032-9592(02)00061-4
31. Kanekar, P. P., Nilegaonkar, S. S., Sarnaik, S. S., Kelkar, A. S. (2002). Optimization of protease activity of alkaliphilic bacteria isolated from an alkaline lake in India. Bioresource Technology, 85(1), 87-93. https://doi.org/10.1016/S0960-8524(02)00018-4
32. Kharadi, A., Chaudhary, K., Patel, F. (2020). Optimization of alkaline protease production by Solibacillus silvestris, isolated from Gir region of Gujarat. Indian Journal of Science and Technology, 13(09), 1065-1077. https://doi.org/10.17485/ ijst/2020/v013i09/149812
33. Lazim, H., Mankai, H., Slama, N., Barkallah, I., Limam, F. (2009). Production and optimization of thermophilic alkaline protease in solid-state fermentation by Streptomyces sp. CN902. Journal of Industrial Microbiology and Biotechnology, 36(4), 531-537. https://doi.org/10.1007/s10295-008-0523-6
34. Mahakhan, P., Apiso, P., Srisunthorn, K., Vichitphan, K., Vichitphan, S., Punyauppa-Path, S., Sawaengkaew, J. (2023). Alkaline protease production from Bacillus gibsonii 6BS15-4 using dairy effluent and its characterization as a laundry detergent additive. Journal of Microbiology and Biotechnology, 33(2), 195. https://doi.org/ 10.4014%2Fjmb.2210.10007
35. Mehrotra, S., Pandey, P. K., Gaur, R., Darmwal, N. S. (1999). The production of alkaline protease by a Bacillus species isolate. Bioresource Technology, 67(2), 201-203. https://doi.org/ 10.1016/S0960-8524(98)00107-2
36. 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 & Biodegradation, 43(1-2), 13-21. https://doi.org/ 10.1016/S0964-8305(98)00061-4
37. Nadeem, M., Qazi, J. I., Baig, S., Syed, Q. U. A. (2008). Effect of medium composition on commercially important alkaline protease production by Bacillus licheniformis N-2. Food Technology and Biotechnology, 46(4), 388-394.
38. Nascimento, W. C. A. D., Martins, M. L. L. (2004). Production and properties of an extracellular protease from thermophilic Bacillus sp. Brazilian Journal of Microbiology, 35, 91-96. https://doi.org/10.1590/S1517-83822004000100015
39. Naveed, M., Nadeem, F., Mehmood, T., Bilal, M., Anwar, Z., Amjad, F. (2021). Protease—a versatile and ecofriendly biocatalyst with multi-industrial applications: an updated review. Catalysis Letters, 151, 307-323. https://doi.org/10.1007/s10562-020-03316-7
40. Olajuyigbe, F. M., Ehiosun, K. I. (2013). Production of thermostable and organic solvent-tolerant alkaline protease from Bacillus coagulans PSB-07 under different submerged fermentation conditions. African Journal of Biotechnology, 12(21). https://doi.org/10.5897/AJB2013.12219
41. Palsaniya, P., Mishra, R., Beejawat, N., Sethi, S., Gupta, B. L. (2012). Optimization of alkaline protease production from bacteria isolated from soil. Journal of Microbiology and Biotechnology Research, 2(6), 858-865.
42. Patel, R. K., Dodia, M. S., Joshi, R. H., Singh, S. P. (2006). Purification and characterization of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp. Process Biochemistry, 41(9), 2002-2009. https://doi.org/ 10.1016/j.procbio.2006.04.016
43. Prakasham, R. S., Rao, C. S., Sarma, P. N. (2006). Green gram husk—an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation. Bioresource Technology, 97(13), 1449-1454. https://doi.org/ 10.1016/j.biortech.2005.07.015
44. Rathod, M. G., Pathak, A. P. (2016). Optimized production, characterization and application of alkaline proteases from taxonomically assessed microbial isolates from Lonar soda lake, India. Biocatalysis and Agricultural Biotechnology, 7, 164-173. https://doi.org/10.1016/ j.bcab.2016.06.002
45. Rekik, H., Jaouadi, N. Z., Gargouri, F., Bejar, W., Frikha, F., Jmal, N., Bejar, S., Jaouadi, B. (2019). Production, purification and biochemical characterization of a novel detergent-stable serine alkaline protease from Bacillus safensis strain RH12. International Journal of Biological Macromolecules, 121, 1227-1239. https://doi.org/ 10.1016/j.ijbiomac.2018.10.139
46. Royter, M., Schmidt, M., Elend, C., Höbenreich, H., Schäfer, T., Bornscheuer, U. T., Antranikian, G. (2009). Thermostable lipases from the extreme thermophilic anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Caldanaerobacter subterraneus subsp. tengcongensis. Extremophiles, 13, 769-783. http://doi.org/10.1007/s00792-009-0265-z
47. Saitou, N., Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4), 406-425. https://doi.org/ 10.1093/oxfordjournals.molbev.a040454
48. Salihi, A., Asoodeh, A., Aliabadian, M. (2017). Production and biochemical characterization of an alkaline protease from Aspergillus oryzae CH93. International Journal of Biological Macromolecules, 94, 827-835. https://doi.org/ 10.1016/j.ijbiomac.2016.06.023
49. Sellami-Kamoun, A., Haddar, A., Ali, N. E. H., Ghorbel-Frikha, B., Kanoun, S., Nasri, M. (2008). Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations. Microbiological Research, 163(3), 299-306. https://doi.org/ 10.1016/j.micres.2006.06.001
50. Sharma, K. M., Kumar, R., Panwar, S., Kumar, A. (2017). Microbial alkaline proteases: Optimization of production parameters and their properties. Journal of Genetic Engineering and Biotechnology, 15(1), 115-126. https://doi.org/ 10.1016/j.jgeb.2017.02.001
51. Silva, C. R. D., Delatorre, A. B., Martins, M. L. L. (2007). Effect of the culture conditions on the production of an extracellular protease by thermophilic Bacillus sp. and some properties of the enzymatic activity. Brazilian Journal of Microbiology, 38, 253-258. http://doi.org/ 10.1590/S1517-83822007000200012
52. Sinha, P., Singh, R. K., Srivastva, R., Sharma, R., Tiwari, S. P. (2013). Characterization and optimization of alkaline protease enzyme produced by soil borne bacteria. Trends in Life Sciences, 2(2), 2319-4731.
53. Tamura, K., Nei, M., Kumar, S. (2004). Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences, 101(30), 11030-11035. http://doi.org/10.1073/pnas.0404206101
54. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28(10), 2731-2739. http://doi.org/ 10.1093/molbev/msr121
55. Thakur, N., Kumar, A., Sharma, A., Bhalla, T. C., Kumar, D. (2018). Purification and characterization of alkaline, thermostable and organic solvent stable protease from a mutant of Bacillus sp. Biocatalysis and Agricultural Biotechnology, 16, 217-224. http://doi.org/ 10.1016/j.bcab.2018.08.005
56. Wilson, P., Remigio, Z. (2012). Production and characterisation of protease enzyme produced by a novel moderate thermophilic bacterium (EP1001) isolated from an alkaline hot spring, Zimbabwe. African Journal of Microbiology Research, 6(27), 5542-5551. http://doi.org/ 10.5897/AJMR11.158
57. Zanphorlin, L. M., Cabral, H., Arantes, E., Assis, D., Juliano, L., Juliano, M. A., Da-Silva, R., Gomes, E., Bonilla-Rodriguez, G. O. (2011). Purification and characterization of a new alkaline serine protease from the thermophilic fungus Myceliophthora sp. Process Biochemistry, 46(11), 2137-2143. http://doi.org/10.1016/ j.procbio.2011.08.014