Substrate Specificity of Bacillus megaterium UСM B-5710 Keratinase
Keywords:Bacillus megaterium UCM B-5710, keratinase, substrate specificity, keratin-containing substrates, α-keratin, β-keratin
The specifics of the processing of livestock and poultry products is that in the process of obtaining the main marketable products, about half the feedstock at various stages of the technological process turns into waste that pollutes the environment. These by-products contain large amounts of the hard-to-digest keratin protein. The use of specific enzymes capable of degrading this protein helps not only to reduce the negative anthropogenic impact on nature but also to obtain valuable hydrolysates that can be used as a fertilizer for plants or a feed additive. The aim of this work was to study the ability of Bacillus megaterium UCM B-5710 to split various keratin-containing substrates: black and white chicken feathers, white turkey feathers, parrot feathers of various colors, sheep wool, pig bristles, and baby hair and nails. Methods. The culture was grown under conditions of submerged cultivation at 40 °C, with a nutrient medium stirring rate of 201 rpm for 6 days. For growth, a basic nutrient medium containing 0.5% defatted chicken feathers or other keratin-containing substrates as sole sources of carbon and nitrogen were used. Keratinase activity was assessed by UV absorption at 280 nm of hydrolysis products of keratin-containing raw materials. Protein was determined by the Lowry method, caseinolytic (total proteolytic) activity was determined by the Anson method modified by Petrova, and amino acid content was determined by the ninhydrin method. The degree of hydrolysis of the substrates was evaluated by the ratio of the initial and final weight of the substrate. Results. It was shown that the synthesis of keratinase by the culture of B. megaterium UCM B-5710 begins from the 6th hour of cultivation. The level of protein and proteolytic activity and the content of amino acids increased throughout the entire period of culture growth. The supernatant of the culture liquid of B. megaterium UCM B-5710 was most effective in splitting white chicken’s and turkey’s feathers, a little slower — feathers of black chicken and blue parrots, as well as wool of white sheep. According to the degree of splitting, the substrates used can be arranged in the following order: white turkey feathers > white chicken feathers > black chicken feathers > blue parrot feathers > white sheep wool > baby nails > pig bristle > baby hair. The study of the effect of feather color on the resistance to decomposition showed that black, blue, and red feathers are more resistant, which coincides with the literature data. Conclusions. B. megaterium UCM B-5710 produces keratinase capable of splitting both α- and β-keratins, however, with different efficiencies and rates.
Qiu J, Wilkens C, Barrett K, Meyer AS. Microbial enzymes catalyzing keratin degradation: Classification, structure, function. Biotechnol Adv. 2020; 44:107607. DOI: https://doi.org/10.1016/j.biotechadv.2020.107607
Anbesaw MS. Bioconversion of keratin wastes using keratinolytic microorganisms to generate value-added products. Int J Biomater. 2022; 2022:2048031. DOI: https://doi.org/10.1155/2022/2048031
Li Q. Progress in Microbial Degradation of Feather Waste. Front Microbiol. 2019; 10:2717. DOI: https://doi.org/10.3389/fmicb.2019.02717
Sypka M, Jodłowska I, Białkowska AM. Keratinases as Versatile Enzymatic Tools for Sustainable Development. Biomolecules. 2021; 11(12):1900. DOI: https://doi.org/10.3390/biom11121900
Latafat, Siddiqui MH, Vimal A, Bhargava P. Biological Degradation of Keratin by Microbial Keratinase for Effective Waste Management and Potent Industrial Applications. Curr Protein Pept Sci. 2021; 22(4):304–312. DOI: https://doi.org/10.2174/1389203722666210215151952
Li ZW, Liang S, Ke Y, Deng GG, Zhang MS. et al. The feather degradation mechanisms of a new Streptomyces sp. isolate SCUT-3. Commun Biol. 2020; 3(1):191. DOI: https://doi.org/10.1038/s42003-020-0918-0
Verma A, Singh H, Anwar MS, Kumar S, Ansari MW, Agrawal S. Production of Thermostable Organic Solvent Tolerant Keratinolytic Protease from Thermoactinomyces sp. RM4: IAA Production and Plant Growth Promotion. Front Microbiol. 2016; 7:1189. DOI: https://doi.org/10.3389/fmicb.2016.01189
Avdiyuk KV, Roy AO. Selection of optimal conditions for cultivation of Bacillus megaterium UCM B-5710 – producer keratinase. Microbiol J. 2021; 83(6):32–40. DOI: https://doi.org/10.15407/microbiolj83.06.032
Nickerson WJ, Noval JJ, Robison RS. Keratinase. I. Properties of the enzyme conjugate elaborated by Streptomyces fradiae. Biochim Biophys Acta. 1963; 77(1):73–86. DOI: https://doi.org/10.1016/0006-3002(63)90470-0
Varbanets LD, Matseliukh EV. Peptidases of microorganisms and methods of their investigations. Kyiv:Naukova Dumka; 2014.
Lowry OH, Rosebrough HJ, Farr AL, Randal RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193(1):265–275. DOI: https://doi.org/10.1016/S0021-9258(19)52451-6
Sharma I, Pranaw K, Soni H, Kango N. Parametrically optimized feather degradation by Bacillus velezensis NCIM 5802 and delineation of keratin hydrolysis by multi-scale analysis for poultry waste management. Sci Rep 2022; 12:17118. DOI: https://doi.org/10.1038/s41598-022-21351-9
Burtt EH Jr, Schroeder MR, Smith LA, Sroka JE, McGraw KJ. Colourful parrot feathers resist bacterial degradation. Biol Lett. 2011; 7(2):214‒216. DOI: https://doi.org/10.1098/rsbl.2010.0716
Gurav RG, Tang J, Jadhav JP. Sulfitolytic and keratinolytic potential of Chryseobacterium sp RBT revealed hydrolysis of melanin containing feathers. 3 Biotech. 2016; 6:145. DOI: https://doi.org/10.1007/s13205-016-0464-0
Wakil SM, Dada MT, Onilude AA. Isolation and Characterization of Keratinase-Producing Bacteria from Poultry Waste. J Pure Appl Microbiol. 2011; 5(2):567-580.
Sharma C, Timorshina S, Osmolovskiy A, Misri J, Singh R. Chicken Feather Waste Valorization Into Nutritive Protein Hydrolysate: Role of Novel Thermostable Keratinase From Bacillus pacificus RSA27. Front Microbiol. 2022; 13:882902. DOI: https://doi.org/10.3389/fmicb.2022.882902
Almahasheer AA, Mahmoud A, El-Komy H, Alqosaibi AI, Aktar S, AbdulAzeez S, Borgio JF. Novel Feather Degrading Keratinases from Bacillus cereus Group: Biochemical, Genetic and Bioinformatics Analysis. Microorganisms. 2022; 10(1):93. DOI: https://doi.org/10.3390/microorganisms10010093
Liaqat I, Ali S, Butt A, Durrani AI, Zafar U, Saleem S, Naseem S, Ahsan F. Purification and Characterization of Keratinase from Bacillus licheniformis dcs1 for Poultry Waste Processing. J Oleo Sci. 2022; 71(5):693-700. DOI: https://doi.org/10.5650/jos.ess21426
Ezeme-Nwafor AC, George-Okafor UO. Studies on Optimal Production of Keratinase by Bacillus cereus 35 Isolated from Feather Dump Sites in Enugu Metropolis, Enugu State, Nigeria. Niger J Microbiol. 2022; 36(1): - 6055 – 6063.
Cai CG, Chen JS, Qi JJ, Yin Y, Zheng XD. Purification and characterization of keratinase from a new Bacillus subtilis strain. J Zhejiang Univ Sci B. 2008; 9(9):713-20. DOI: https://doi.org/10.1631/jzus.B0820128
Gunderson AR, Frame AM, Swaddle JP, ForsythJ MH. Resistance of melanized feathers to bacterial degradation: is it really so black and white? Avian Biol. 2008; 39: 539‒545. DOI: https://doi.org/10.1111/j.0908-8857.2008.04413.x
How to Cite
Copyright (c) 2023 Mikrobiolohichnyi Zhurnal
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.