PERTANIKA JOURNAL OF SOCIAL SCIENCES AND HUMANITIES

 

e-ISSN 2231-8534
ISSN 0128-7702

Home / Regular Issue / JSSH Vol. 45 (4) Nov. 2022 / JTAS-2451-2022

 

Isolation, Characterization, and Optimization of Keratinase from Bacillus cereus BRAW_KM

Raden Lukas Martindro Satrio Ari Wibowo, Atiqa Rahmawati and Ragil Yuliatmo

Pertanika Journal of Social Science and Humanities, Volume 45, Issue 4, November 2022

DOI: https://doi.org/10.47836/pjtas.45.4.07

Keywords: Bacillus cereus, characterization, isolation, keratinase enzyme, optimization

Published on: 4 November 2022

Indonesia possesses tremendous marine resources. Therefore, their marine products are appropriate for exploration. In the prior study, bacteria generating keratinase enzyme have isolated from local fish market trash. The keratinase may hydrolyze keratin on the skin. Surrounding parameters, such as temperature, pH, and incubation duration, are the factors affecting the activity of the enzyme. This study aims to isolate and characterize keratinase, and optimize its production. The enzyme from Bacillus cereus BRAW_KM was the main material utilized in this research. First, the keratinolytic bacterium was isolated and investigated the properties of keratinase using native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. Then, the ideal conditions of keratinase synthesis were adjusted by temperature, pH, and incubation time on enzyme activity. Of 10 isolations discovered, one isolate shows the potential as a keratinolytic bacterium, which tends to behave like Bacillus sp. The molecular weights of keratinase were 130 kDa and 95 kDa. The optimum keratinase enzyme activity from B. cereus BRAW_KM was at 29 °C, pH 9, and 90 minutes of incubation.

  • Balakumar, S., Mahesh, N., Arunkumar, M., Sivakumar, R., & Hemambujavalli, V. (2013). Optimization of keratinase production by Keratinolytic organisms under submerged fermentation. International Journal of PharmTech Research, 5(3), 1294–1300.

  • Bhari, R., Kaur, M., & Singh, R. S. (2019). Thermostable and halotolerant keratinase from Bacillus aerius NSMk2 with remarkable dehairing and laundary applications. Journal of Basic Microbiology, 59(6), 555–568. https://doi.org/10.1002/jobm.201900001

  • Bergey, D. H., & Gibbons, N. E. (1974). Bergey’s manual of determinative bacteriology (8th ed.). Williams & Wilkins Company.

  • Dettmer, A., Záchia Ayub, M. A., & Gutterres, M. (2011). Hide unhairing and characterization of commercial enzymes used in leather manufacture. Brazilian Journal of Chemical Engineering, 28(3), 373–380. https://doi.org/10.1590/S0104-66322011000300003

  • Dettmer, A., Cavalheiro, J. C., Cavalli, É., Rossi, D. M., de Souza Gusatti, C., Záchia Ayub, M. A., & Gutterres, M. (2012). Optimization of the biotechnological process for hide unhairing in substitution of toxic sulfides. Chemical Engineering and Technology, 35(5), 803–810. https://doi.org/10.1002/ceat.201100350

  • Dettmer, A., Cavalli, É., Záchia Ayub, M. A., & Gutterres, M. (2013). Environmentally friendly hide unhairing: Enzymatic hide processing for the replacement of sodium sulfide and deliming. Journal of Cleaner Production, 47, 11–18. https://doi.org/10.1016/j.jclepro.2012.04.024

  • Food and Agriculture Organization of the United Nations. (2019). Fishery and aquaculture country profiles: Indonesia. FAO. https://www.fao.org/fishery/en/facp/101/en

  • Gegeckas, A., Šimkutė, A., Gudiukaitė, R., & Čitavičius, D. J. (2018). Characterization and application of keratinolytic paptidases from Bacillus spp.. International Journal of Biological Macromolecules, 113, 1206–1213. https://doi.org/10.1016/j.ijbiomac.2018.03.046

  • Gessesse, A., Hatti-Kaul, R., Gashe, B. A., & Mattiasson, B. (2003). Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. Enzyme and Microbial Technology, 32(5), 519–524. https://doi.org/10.1016/S0141-0229(02)00324-1

  • Gupta, S., Nigam, A., & Singh, R (2015). Purification and characterization of a Bacillus subtilis keratinase and its prospective application in feed industry. Acta Biologica Szegediensis, 59(2), 197–204.

  • Hiol, A., Jonzo, M. D., Druet, D., & Comeau, L. (1999). Production, purification and characterization of an extracellular lipase from Mucor hiemalis f. hiemalis. Enzyme and Microbial Technology, 25(1-2), 80–87. https://doi.org/10.1016/S0141-0229(99)00009-5

  • Hoq, M. M., Siddiquee, K. A. Z., Kawasaki, H., & Seki, T. (2005). Keratinolytic activity of some newly isolated Bacillus species. Journal of Biological Sciences, 5(2), 193–200. https://doi.org/10.3923/jbs.2005.193.200

  • Kandasamy, N., Velmurugan, P., Sundarvel, A., Raghava, R. J., Bangaru, C., & Palanisamy, T. (2012). Eco-benign enzymatic dehairing of goatskins utilizing a protease from a Pseudomonas fluorescens species isolated from fish visceral waste. Journal of Cleaner Production, 25, 27–33. https://doi.org/10.1016/j.jclepro.2011.12.007

  • Kumar, A. G., Swarnalatha, S., Gayathri, S., Nagesh, N., & Sekaran, G. (2008). Characterization of an alkaline active - Thiol forming extracellular serine keratinase by the newly isolated Bacillus pumilus. Journal of Applied Microbiology, 104(2), 411–419. https://doi.org/10.1111/j.1365-2672.2007.03564.x

  • Laba, W., & Rodziewicz, A. (2014). Biodegradation of hard keratins by two Bacillus strains. Jundishapur Journal of Microbiology, 7(2), e8896. https://doi.org/10.5812%2Fjjm.8896

  • Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. https://doi.org/10.1038/227680a0

  • Lin, X., Shih, J. C. H., & Swaisgood, H. E. (1996). Hydrolysis of feather keratin by immobilized keratinase. Applied and Environmental Microbiology, 62(11), 4273–4275. https://doi.org/10.1128/aem.62.11.4273-4275.1996

  • Mazotto, A. M., Coelho, R. R. R., Cedrola, S. M. L., de Lima, M. F., Couri, S., de Souza, E. P., & Vermelho, A. B. (2011). Keratinase production by three Bacillus spp. using feather meal and whole feather as substrate in a submerged fermentation. Enzyme Research, 2011, 523780. https://doi.org/10.4061/2011/523780

  • Mazotto, A. M., de Melo, A. C. N., Macrae, A., Rosado, A. S., Peixoto, R., Cedrola, S. M. L., Couri, S., Zingali, R. B., Villa, A. L. V., Rabinovitch, L., Chaves, J. Q., & Vermelho, A. B. (2011). Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp.. World Journal of Microbiology and Biotechnology, 27, 1355–1365. https://doi.org/10.1007/s11274-010-0586-1

  • Mechri, S., Kriaa, M., Ben, M., Berrouina, M. B. E., Benmrad, M. O., Jaouadi, N. Z., Rekik, H., Bouacem, K., Bouanane-Darenfed, A., Chebbi, A., Sayadi, S., Chamkha, M., Bejar, S., & Jaouadi, B. (2017). Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R. International Journal of Biological Macromolecules, 101, 383–397. https://doi.org/10.1016/j.ijbiomac.2017.03.051

  • Mousavi, S., Salouti, M., Shapoury, R., & Heidari, Z. (2013). Optimization of keratinase production for feather degradation by Bacillus subtilis. Jundishapur Journal of Microbiology, 6(8), 7160. https://doi.org/10.5812/jjm.7160

  • Nayaka, S., Babu, K. G., & Vidyasagar, G. M. (2013). Purification and characterization of keratinase from hair-degrading Streptomyces albus. International Jornal of Bioassays, 2(3), 599-604.

  • Ogino, H., Otsubo, T., & Ishikawa, H. (2008). Screening, purification, and characterization of a leather-degrading protease. Biochemical Engineering Journal, 38(2), 234–240. https://doi.org/10.1016/j.bej.2007.07.008

  • Sattayasai, N. (2012). Protein purification. In D. Ekinci (Ed.), Chemical biology. IntechOpen. https://doi.org/10.5772/35425

  • Selvam, K., Vishnupriya, B., & Yamuna, M. (2013). Isolation and description of keratinase producing marine actinobacteria from South Indian Coastal Region. African Journal of Biotechnology, 12(1), 19–26. https://doi.org/10.5897/AJB12.2428

  • Sharma, R., & Gupta, R. (2010). Substrate specificity characterization of a thermostable keratinase from Pseudomonas aeruginosa KS-1. Journal of Industrial Microbiology and Biotechnology, 37(8), 785–792. https://doi.org/10.1007/s10295-010-0723-8

  • Sivakumar, T., Shankar, T., & Ramasubramanian, V. (2012). Purification properties of Bacillus thuringiensis TS2 keratinase enzyme. American-Eurasian Journal of Agriculture and Environtment Science, 12(12), 1553-1557.

  • Tamersit, S., & Bouhidel, K. E. (2020). Treatment of tannery unhairing wastewater using carbon dioxide and zinc cations for greenhouse gas capture, pollution removal and water recycling. Journal of Water Process Engineering, 34, 101120. https://doi.org/10.1016/j.jwpe.2019.101120

  • Tatineni, R., Doddapaneni, K. K., Potumarthi, R. C., Vellanki, R. N., Kandathil, M. T., Kolli, N., & Mangamoori, L. N. (2008). Purification and characterization of an alkaline keratinase from Streptomyces sp.. Bioresource Technology, 99(6), 1596–1602. https://doi.org/10.1016/j.biortech.2007.04.019

  • Tran, N., Rodriguez, U. P., Chan, C. Y., Phillips, M. J., Mohan, C. V., Henriksson, P. J. G., Koeshendrajana, S., Suri, S., & Hall, S. (2017). Indonesian aquaculture futures: An analysis of fish supply and demand in Indonesia to 2030 and role of aquaculture using the AsiaFish model. Marine Policy, 79, 25–32. https://doi.org/10.1016/j.marpol.2017.02.002

  • Wang, R. U. I., Min, C., Haiming, C., & Li, Z. (2009). Enzyme unhairing - An eco-friendly biotechnological process. Journal of the Society Leather Technologists and Chemists, 93(2), 51–55.

  • Wibowo, R. L. M. S. A., & Yuliatmo. R. (2020). Characterization and production optimization of keratinase from three Bacillus strains. Leather and Footwear Journal, 20(4), 375-384. https://doi.org/10.24264/lfj.20.4.4

  • Wibowo, R. L. M. S. A., Bachruddin, Z., Fitriyanto, N. A., Nakagawa, T., Hayakawa, T., & Pertiwiningrum, A. (2017a). Screening and characterization of keratinolytic bacteria from puffer fish skin waste. Pakistan Journal of Nutrition, 16(7), 488–496. https://doi.org/10.3923/pjn.2017.488.496

  • Wibowo, R. L. M. S. A., Bachruddin, Z., Fitriyanto, N. A., Nakagawa, T., Hayakawa, T., & Pertiwiningrum, A. (2017b). Isolation of bacteria producing enzyme collagenase from waste of pufferfish (Arothon reticularis) skin. https://journal.ugm.ac.id/istapproceeding/article/view/29845/17969

  • Wilson, K., & Walker, J. (Eds.) (2010). Principles and techniques of biochemistry and molecular biology (7th ed.). Cambridge University Press.

  • Yuliatmo, R., Fitriyanto, N. A., Bachruddin, Z., Supadmo., Jamhari., & Erwanto, Y. (2017). Increasing of angiotensin converting enzyme inhibitory derived from Indonesian native chicken leg protein using Bacillus cereus protease enzyme. International Food Research Journal, 24(4), 1799–1804.