Потенциал использования штаммов Bacillus subtilis A5.3 и Bacillus licheniformis T7 в гидролизе коллагенсодержащих отходов сельского хозяйства
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Abstract
Исследование посвящено потенциалу Bacillus subtilis A5.3 и Bacillus licheniformis T7 в качестве источников протеолитических ферментов с коллагеназной активностью для биотехнологической переработки коллагенсодержащих отходов сельского хозяйства. Были проведены выделение, идентификация и характеризация этих бактериальных штаммов с использованием морфологических, биохимических и молекулярно-генетических методов. Протеолитическая активность оценивалась на казеине и желатине в качестве субстратов. Оптимальные условия для активности ферментов были определены как 70°C и pH 10,0 для B. subtilis A5.3 и 60°C и pH 9,0 для B. licheniformis T7. Исследование влияния ионов металлов, детергентов и ингибиторов на активность ферментов выявило значительные различия между двумя штаммами: B. subtilis A5.3 продемонстрировал более высокую чувствительность к ионам Mn²⁺, Cd²⁺ и Mg²⁺, тогда как B. licheniformis T7 показал большую устойчивость к этим металлам. Зимографический анализ подтвердил наличие нескольких активных протеаз в ферментных экстрактах обоих штаммов с молекулярными массами от 20 до 150 кДа. Наблюдаемая активность гидролиза желатина свидетельствует о наличии коллагеназоподобных протеаз, что подчеркивает промышленный потенциал этих штаммов в гидролизе коллагенсодержащих отходов. Полученные результаты подтверждают пригодность B. subtilis A5.3 и B. licheniformis T7 в качестве перспективных кандидатов для разработки устойчивых методов утилизации коллагенсодержащих отходов.
Article Details
Accepted 2025-09-30
Published 2024-12-30
References
Szucs, M., Angulo, M., Costa, C., Márquez, M.C. Meat waste valorization through protein hydrolysis using different types of proteases // Recent Progress in Materials. – 2021. – Vol. 03(04). – P. 045. https://doi.org/10.21926/rpm.2104045.
Morimura, S., Nagata, H., Uemura, Y., Fahmi, A., Shigematsu, T., Kida, K. Development of an effective process for utilization of collagen from livestock and fish waste // Process Biochemistry. – 2002. – Vol. 37(12). – P. 1403-12. https://doi.org/10.1016/S0032-9592(02)00024-9.
Aspevik, T., Oterhals, Å., Rønning, S.B., Altintzoglou, T., Wubshet, S.G., Gildberg, A., et al. Valorization of Proteins from Co- and By-Products from the Fish and Meat Industry. In: Lin CSK, editor. Chemistry and Chemical Technologies in Waste Valorization. Cham: Springer International Publishing. – 2018. – P. 123-50.
Ricard-Blum, S. The collagen family // Cold Spring Harbor perspectives in biology. – 2011. – Vol. 3(1). – P. a004978. https://doi.org/10.1101/cshperspect.a004978.
Pardo, A., Selman, M. MMP-1: the elder of the family // The international journal of biochemistry & cell biology. – 2005. – Vol. 37(2). – P. 283-8. https://doi.org/10.1016/j.biocel.2004.06.017.
Cavello, I.A., Cavalitto, S.F., Hours, R.A. Biodegradation of a Keratin Waste and the Concomitant Production of Detergent Stable Serine Proteases from Paecilomyces lilacinus // Applied biochemistry and biotechnology. – 2012. – Vol. 167(5). – P. 945-58. https://doi.org/10.1007/s12010-012-9650-7.
Dai, Z., Wu, Z., Jia, S., Wu, G. Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection // Journal of Chromatography B. – 2014. – Vol. 964. – P. 116-27. https://doi.org/10.1016/j.jchromb.2014.03.025.
Tavano, O.L. Protein hydrolysis using proteases: An important tool for food biotechnology. Journal of Molecular Catalysis B // Enzymatic. – 2013. – Vol. 90. – P. 1-11. https://doi.org/10.1016/j.molcatb.2013.01.011.
Brandelli, A. Bacterial Keratinases: Useful Enzymes for Bioprocessing Agroindustrial Wastes and Beyond // Food and Bioprocess Technology. – 2008. – Vol. 1(2). – P. 105-16. https://doi.org/10.1007/s11947-007-0025-y.
Paul, T., Jana, A., Das, A., Mandal, A., Halder, S.K., Das Mohapatra, P.K., et al. Smart cleaning-in-place process through crude keratinase: an eco-friendly cleaning techniques towards dairy industries // Journal of Cleaner Production. – 2014. – Vol. 76. – P. 140-53. https://doi.org/10.1016/j.jclepro.2014.04.028.
Bracho, G.E., Haard, N.F. Idettification of two matrix metalloproteinase in the skeletal muscle of pacific rockfish (Sebaster sp.) // Journal of Food Biochemistry. – 1995. – Vol. 19(4). – P. 299-319. https://doi.org/10.1111/j.1745-4514.1995.tb00536.x.
Kristjánsson, M.M., Gudmundsdóttir, S., Fox, J.W., Bjarnason, J.B. Characterization of a collagenolytic serine proteinase from the Atlantic cod (gadus morhua). Comparative Biochemistry and Physiology Part B // Biochemistry and Molecular Biology. – 1995. – Vol. 110(4). – P. 707-17. https://doi.org/10.1016/0305-0491(94)00207-B.
Kolodziejska I., Sikorski Z.E. Neutral and alkaline muscle proteinases of marine fish and invertebrates a review // Journal of Food Biochemistry. – 1996. – Vol. 20(3). – P. 349-64. https://doi.org/10.1111/j.1745-4514.1996.tb00561.x.
Oliveira, Vd.M., da Cunha, M., Assis, C., Nascimento, T., Herculano, P., Cavalcanti, M., et al. Fish collagenases and its industrial applications // Pubvet. – 2017. – Vol . 11(3). – P. 243-55. https://doi.org/10.22256/PUBVET.V11N3.243-255.
Eizen, A.Z., Jeffrey, J.J. An extractable collagenase from crustacean hepatopancreas. Biochimica et Biophysica Acta (BBA) // Enzymology. – 1969. – Vol. 191(3). – P. 517-26. https://doi.org/10.1016/0005-2744(69)90345-3.
Homaei, A., Lavajoo, F., Sariri, R. Development of marine biotechnology as a resource for novel proteases and their role in modern biotechnology // International Journal of Biological Macromolecules. – 2016. – Vol. 88. – P. 542-52. https://doi.org/10.1016/j.ijbiomac.2016.04.023.
Feng, L., Qiao, Y., Zou, Y., Huang, M., Kang, Z., Zhou, G. Effect of Flavourzyme on proteolysis, antioxidant capacity and sensory attributes of Chinese sausage // Meat Science. – 2014. – Vol. 98(1). – P. 34-40. https://doi.org/10.1016/j.meatsci.2014.04.001.
Yang, H., Qu, J., Zou, W., Shen, W., Chen, X. An overview and future prospects of recombinant protein production in Bacillus subtilis // Appl Microbiol Biotechnol. – 2021. – Vol. 105(18). – P. 6607-26. https://doi.org/10.1007/s00253-021-11533-2.
Lajis, A.F.B. Biomanufacturing process for the production of bacteriocins from Bacillaceae family // Bioresources and Bioprocessing. – 2020. – Vol. 7(1). – P. 1-26. https://doi.org/10.1186/s40643-020-0295-z.
Arora, G., Patil, A., Hooshanginezhad, Z., Fritz, K., Salavastru, C., Kassir, M., et al. Cellulite: Presentation and management // Journal of Cosmetic Dermatology. – 2022. – Vol. 21(4). – P. 1393-401. https://doi.org/10.1111/jocd.14815.
Costas, B., Coleman, S., Kaufman, G., James, R., Cohen, B., Gaston, R.G. Efficacy and safety of collagenase clostridium histolyticum for Dupuytren disease nodules: a randomized controlled trial // BMC Musculoskeletal Disorders. – 2017. – Vol. 18(1). – P. 374. https://doi.org/10.1186/s12891-017-1713-z.
Gemechu, G., Masi, C., Tafesse, M., Kebede, G. A review on the bacterial alkaline proteases // J. Xidian Univ. – 2020. – Vol. 14(11). – P. 264-274. https://doi.org/10.37896/jxu14.11/022.
Gimenes, N.C., Silveira, E., Tambourgi, E.B. An Overview of Proteases: Production, Downstream Processes and Industrial Applications // Separation & Purification Reviews. – 2021. – Vol. 50(3). – P. 223-243. https://doi.org/10.1080/15422119.2019.1677249.
Contesini, F.J., Melo, R.R., Sato, H.H. An overview of Bacillus proteases: from production to application // Critical reviews in biotechnology. – 2018. – Vol. 38(3). – P. 321-334. https://doi.org/10.1080/07388551.2017.1354354.
Danilova, I., Sharipova, M. The practical potential of bacilli and their enzymes for industrial production // Frontiers in microbiology. – 2020. – Vol. 11. – P. 1782. https://doi.org/10.3389/fmicb.2020.01782.
Cai, D., Rao, Y., Zhan, Y., Wang, Q., Chen, S. Engineering Bacillus for efficient production of heterologous protein: current progress, challenge and prospect // Journal of Applied Microbiology. – 2019. – Vol. 126(6). – P. 1632-42. https://doi.org/10.1111/jam.14192.
Hoppe, I.J., Brandstetter, H., Schönauer, E. Biochemical characterisation of a collagenase from Bacillus cereus strain Q1 // Scientific Reports. – 2021. – Vol. 11(1). – P. 4187. https://doi.org/10.1038/s41598-021-83744-6.
Samritphol, W., Sumpavapol, P., Tangwatcharin, P., Sorapukdee, S. Hydrolytic properties of crude protease from Bacillus subtilis subsp. subtilis M13 // J. Agric. Sci. Technol. – 2019. – Vol. 5(6). – P. 1011-20.
Wang, Y., Li, X., Zhang, Z., Ding, S., Jiang, H., Li, J., et al. Simultaneous determination of nitroimidazoles, benzimidazoles, and chloramphenicol components in bovine milk by ultra-high performance liquid chromatography–tandem mass spectrometry // Food Chemistry. – 2016. – Vol. 192. – P. 280-7. https://doi.org/10.1016/j.foodchem.2015.07.033.
Savita, K., Arachana, P. Production of collagenase by Bacillus KM369985 isolated from leather sample // Int. J. Res. Biosciences. – 2015. – Vol. 4(4). – P. 81-87.
Abfalter, C.M., Schönauer, E., Ponnuraj, K., Huemer, M., Gadermaier, G., Regl, C., et al. Cloning, purification and characterization of the collagenase cola expressed by Bacillus cereus ATCC 14579 // Plos One. – 2016. – Vol. 11(9). – P. e0162433. https://doi.org/10.1371/journal.pone.0162433.
Liu, L., Ma, M., Cai, Z., Yang, X., Wang, W. Purification and properties of a collagenolytic protease produced by Bacillus cereus MBL13 strain // Food Technol Biotechnol. – 2010. – Vol. 48(2). – P. 151.
Song, Y., Fu, Y., Huang, S., Liao, L., Wu, Q., Wang, Y., et al. Identification and antioxidant activity of bovine bone collagen-derived novel peptides prepared by recombinant collagenase from Bacillus cereus // Food Chemistry. – 2021. – Vol. 349. – P. 129143. https://doi.org/10.1016/j.foodchem.2021.129143.
Makinen, K.K., Makinen, P.L. Purification and properties of an extracellular collagenolytic protease produced by the human oral bacterium Bacillus cereus (strain Soc 67) // J. Biol. Chem. – 1987. – Vol. 262(26). – P. 12488-95. https://doi.org/10.1016/S0021-9258(18)45232-5.
Wu, Q., Li, C., Li, C., Chen, H., Shuliang, L. Purification and Characterization of a Novel Collagenase from Bacillus pumilus Col-J // Appl. Biochem. Biotechnol. – 2009. – Vol. 160(1). – P. 129. https://doi.org/10.1007/s12010-009-8673-1.
Zhang, X.-X., Li, Y., Wang, S.-Y., Wang, Y.-Y., Du, K.-L., Xu, J.-Y., Lei, L.-S., Feng, X., Liang, X.-Y., Ruan, H.-H. Expression, purification and enzymatic characterization of ColR75E collagenase of Bacillus cereus R75E // Chin. Biotechnol. – 2015. – Vol. 35(10). – P. 44-52. https://doi.org/10.13523/j.cb.20151007.
Okamoto, M., Yonejima, Y., Tsujimoto, Y., Suzuki, Y., Watanabe, K. A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1 // Appl. Microbiol. Biotechnol. – 2001. – Vol. 57(1). – P. 103-8. https://doi.org/10.1007/s002530100731.
Zhu, Y., Wang, L., Zheng, K., Liu, P., Li, W., Lin, J., et al. Optimized recombinant expression and characterization of collagenase in Bacillus subtilis WB600 // Fermentation. – 2022. – Vol. 8(9). – P. 449. https://doi.org/10.3390/fermentation8090449.
Pequeno, A.C.L., Arruda, A.A., Silva, D.F., Duarte Neto, J.M.W., Silveira Filho, V.M., Converti, A., et al. Production and characterization of collagenase from a new Amazonian Bacillus cereus strain // Preparative Biochemistry & Biotechnology. – 2019. – Vol. 49(5). – P. 501-9. https://doi.org/10.1080/10826068.2019.1587627.
Nagano, H., To, K.A. Purification of Collagenase and Specificity of Its Related Enzyme from Bacillus subtilis FS-2 // Biosci. Biotechnol. Biochem. – 2000. – Vol. 64(1). – P. 181-3. https://doi.org/10.1271/bbb.64.181.
Kawahara, H., Kusumoto, M., Obata, H. Isolation and characterization of a new-type of collagenase producing bacterium, Bacillus-alvei DC-1 // Biosci. Biotechnol. Biochem. -1993. – Vol. 57(8). – P. 1372-3. https://doi.org/10.1271/bbb.57.1372.
Asdornnithee, S., Akiyama, K., Sasaki, T., Takata, R. Isolation and characterization of a collagenolytic enzyme from Bacillus licheniformis N22 // J. Ferment. Bioeng. – 1994. – Vol. 78(4). – P. 283-7. https://doi.org/10.1016/0922-338X(94)90358-1.
Kembhavi, A.A., Kulkarni, A., Pant, A. Salt-tolerant and thermostable alkaline protease from Bacillus subtilis NCIM no. 64 // Appl. Biochem. Biotechnol. – 1993. – Vol. 38(1-2). – P. 83-92. https://doi.org/10.1007/bf02916414.
Silva, C.Rd., Delatorre, A.B., Martins, M.L.L. 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. – 2007. – Vol. 38. – P. 253-8. https://doi.org/10.1590/S1517-83822007000200012.
Farhadian, S., Asoodeh, A., Lagzian, M. Purification, biochemical characterization and structural modeling of a potential htrA-like serine protease from Bacillus subtilis DR8806 // Journal of Molecular Catalysis B: Enzymatic. – 2015. – Vol. 115. – P. 51-8. https://doi.org/10.1016/j.molcatb.2015.02.001.
Adinarayana, K., Ellaiah, P., Prasad, D.S. Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11 // AAPS PharmSciTech. – 2003. – Vol. 4(4). – P. E56. https://doi.org/10.1208/pt040456.
Sundarrajan, S., Parambath, S., Suresh, S., Rao, S., Padmanabhan, S. Novel properties of recombinant Sso7d-Taq DNA polymerase purified using aqueous two-phase extraction: Utilities of the enzyme in viral diagnosis // Biotechnol. Rep. (Amst). – 2018. – Vol. 19. – P. e00270. https://doi.org/10.1016/j.btre.2018.e00270.
Verma, A., Singh, H., Anwar, S., Chattopadhyay, A., Tiwari, K.K., Kaur, S., et al. Microbial keratinases: industrial enzymes with waste management potential // Crit. Rev. Biotechnol. – 2017. – Vol. 37(4). – P. 476-91. https://doi.org/10.1080/07388551.2016.1185388.
Prakash, P., Jayalakshmi, S.K., Sreeramulu, K. Purification and characterization of extreme alkaline, thermostable keratinase, and keratin disulfide reductase produced by Bacillus halodurans PPKS-2 // Appl. Microbiol. Biotechnol. – 2010. – Vol. 87(2). – P. 625-33. https://doi.org/10.1007/s00253-010-2499-1.
Tiwary, E., Gupta, R. Medium optimization for a novel 58 kDa dimeric keratinase from Bacillus licheniformis ER-15: biochemical characterization and application in feather degradation and dehairing of hides // Bioresour. Technol. – 2010. – Vol. 101(15). – P. 6103-10. https://doi.org/10.1016/j.biortech.2010.02.090.
Mazotto, A.M., de Melo, A.C., Macrae, A., Rosado, A.S., Peixoto, R., Cedrola, S.M., et al. Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp // World J. Microbiol. Biotechnol. – 2011. – Vol. 27(6). – P. 1355-65. https://doi.org/10.1007/s11274-010-0586-1.
Kumar, A.G., Swarnalatha, S., Gayathri, S., Nagesh, N., Sekaran, G. Characterization of an alkaline active-thiol forming extracellular serine keratinase by the newly isolated Bacillus pumilus // J. Appl. Microbiol. – 2008. – Vol. 104(2). – P. 411-9. https://doi.org/10.1111/j.1365-2672.2007.03564.x.
Suh, H.J., Lee, H.K. Characterization of a keratinolytic serine protease from Bacillus subtilis KS-1 // J. Protein Chem. – 2001. – Vol. 20(2). – P. 165-9. https://doi.org/10.1023/a:1011075707553.
Younes, G., Shahbazi, M., Rasoul-Amini, S., Kargar, M., Safari, A., Kazemi, A., et al. Identification and characterization of feather-degrading bacteria from keratin-rich wastes // Ann. Microbiolog. – 2012. – Vol. 62. – P. 737-744. https://doi.org/10.1007/s13213-011-0313-7.
Riffel, A., Lucas, F., Heeb, P., Brandelli, A. Characterization of a new keratinolytic bacterium that completely degrades native feather keratin // Arch. Microbiol. – 2003. – Vol. 179(4). – P. 258-65. https://doi.org/10.1007/s00203-003-0525-8.