Purification of Wickerhamomyces anomalus  Keratinase and Its Prospective Application in Poultry Feed Industries

D. A. Aina; C. O. Ezeamagu; S. T. Akindele; A. O. Aleshinloye

doi:10.53704/fujnas.v10i1.345

Authors

D. A. Aina Department of Microbiology, Babcock University, Ilishan-Remo, Ogun State, Nigeria.
C. O. Ezeamagu Department of Microbiology, Babcock University, Ilishan-Remo, Ogun State, Nigeria.
S. T. Akindele Department of Microbiology, Babcock University, Ilishan-Remo, Ogun State, Nigeria.
A. O. Aleshinloye Chemistry Unit, Department of Basic Sciences, Babcock University, Ilishan-Remo, Ogun State, Nigeria.

DOI:

https://doi.org/10.53704/fujnas.v10i1.345

Abstract

Animal wastes emanating from cow horns, hooves and feathers are keratinous in nature which can only be degraded by keratinolytic microorganisms. Consequently, the pollution resulting from the accumulation of these wastes in response to growing livestock demand is posing a significant threat to human health and the environment. This study was carried out using cow hooves as the substrate for the production of keratinase from fungal identified as Wickerhamomyces anomalus 9 (18S rDNA gene sequencing) was isolated from soil rich hooves using basal salt agar medium and potato dextrose agar. The keratinase of the isolate was assessed using skim milk agar and the enzyme was produced by solid-state fermentation. The crude enzyme was purified using ammonium sulphate precipitation, ion exchange and gel-filtration chromatography. The specific activity of the enzyme was 0.29 U/mg with a yield of 45% and a 7.25 purification fold. The optimal pH and temperature of the enzyme were 8.0 and 60 ^oC respectively. The enzyme was observed to be thermo-stable at 50 ^oC between for 30 minutes. The kinetics revealed that the V_max was 0.384 U/min with K_m 86.95 mg/ml. The native molecular weight of the enzyme was found to be 34 KDa. There were significant differences at 95% confidence with poultry feed treated with Wickerhamomyces anomalus keratinase in moisture, ash content, crude fibre, crude fat, nitrogen content, crude protein and carbohydrate compare to the untreated feed. These results suggest an environment-friendly approach for biodegradation of cow hooves wastes for the production of keratinases, animal waste management as well as a promising tool for chicken feed additives.

Keywords: Biodegradation, Chromatography, Cow hooves, Keratinase, Pollution

References

Abdul Gafar, A., Khayat, M. E., Ahmad, S. A., Yasid, N. A. & Shukor, M. Y. (2020). Response Surface Methodology for the Optimization of Keratinase Production in Culture Medium Containing Feathers by Bacillus sp. UPM- AAG1 Catalysts 10: 848. doi:10.3390/catal10080848

Adefisoye, S., & Abdulwasiu, S. (2018). Production of Glucoamylase by Aspergillus niger in Solid State Fermentation. 12. 7-11. doi:10.5829/idosi.abr.2018.07.11.

Al-Ghanim, K., Al-Thobaiti, A., Al-Balawi, H.F., Alkahem, A.Z., & Mahboob, S. (2017). Effects of Replacement of Fishmeal with other Alternative Plant Sources in the Feed on Proximate Composition of Muscle, Liver and Ovary in Tilapia (Oreochromis nioloticus). Brazilian Archives of Biology and Technology, 60,e17160376. doi: https://dx.doi.org/10.1590/1678- 4324 2017160376

Ali, T. H., Ali, N. A., & Mohamed, L. A. (2011). Production, purification and some properties of extracellular keratinase from feather degradation by Aspergillusoryzae NRRL-447. J. Appl Sci. Environ Sanit 6:123-36.

AOAC (2006). Official Methods of Analysis. 18th Edition, Association of Official Analytical Chemists, Gaithersburgs, M.D.

Bach, E., Sant’Anna, V., Daroit, D. J., Corrêa, A. P. F., Segalin, J., and Brandelli, A. (2012). Production, one-step purification, and characterization of a keratinolytic protease from Serratia marcescens P3. Process Biochem. 47, 2455–2462. doi: 10.1016/j.procbio.2012.10.007

Barman, N.C., Zohora, F.T., Das, K.C., Mowla, M.G., Banu, N.K., Salimullah, M & Hashem, A. (2017). Production, partial optimization and characterization of keratinase enzyme by Arthrobacter sp. NFH5 isolated from soil samples. AMB Express.7:18

Baweja, M., Tiwari, R., Singh, P.K., Nain, L., & Shukla, P. (2016). An alkaline protease from Bacillus pumilus MP 27: functional analysis of its binding model toward its application as detergent additive, Front. Microbiol. 7: 1195. doi:http://dx.doi.org/10.3389/fmicb.2016.01195

Bradford, M.M. (1979). A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal-Biochemistry 72, 248-54.

Cai, S.B., Huang, Z.H., Zhang, X.Q. et al., (2011). Appl Biochem Biotechnol 163: 112. https://doi.org/10.1007/s12010-010-9021-1

Couri, S., Terzi, S., Pinto, G., Freitas, S., & De Costa, A.C. (2000). Hydrolytic enzyme production in solid-state fermentation by Aspergillus niger 3T5B8. Process Biochemistry 36:255-261. https://doi.org/10:1016/500329592(00)00209-0

Esawy, M.A. (2007). Isolation and Partial Characterization of Extracellular Keratinase from a Novel Mesophilic Streptomyces albus AZA. Res. J. Agric. and Biol. Sci., 3(6), 808-817.

Fang, Z., Zhang, J., Liu, B., & Jiang, L. (2014). Cloning, heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1.Process Biochem. 49, 647–654.

Fontoura, R., Daroit, D.J., Correa, A.P., Meira, S.M., Mosquera, M. & Brandelli, A. (2014). Production of feather hydrolysates with antioxidant, angiotensin-I converting enzyme and dipeptidyl peptidase-IV-inhibitory activities. New Biotechnol. 31:506–513.

Gafar, A., Khayat, B, Mohd Ezuan., Ahmad, Siti Aqlima., Yasid, N., & Shukor, Y. (2020). Response Surface Methodology for the Optimization of Keratinase Production in Culture Medium Containing Feathers by Bacillus sp. UPM-AAG1. Catalysts. 10. 848. doi: 10.3390/catal10080848.

Godbole, S., Decker, S.R., Nieves, R.A., Adney, W.S., Vinzant, T.B., Baker, J.O., Thomas, S.R., & Himmel, M.E. (1999). Cloning and expression of Trichoderma reesei cellobiohydrolase I in Pichia pastoris. Biotechnological Progress.15:828–833. doi: 10.1021/bp9901116.

Huang, Y., Lezyk, M., Herbst, F.A., Busk, P.K & Lange, L. (2020). Novel keratinolytic enzymes, discovered from a talented and efficient bacterial keratin degrader. Sci Rep 10, 10033 (2020). https://doi.org/10.1038/s41598-020-66792-2

Isiaka, A., & Agbaje, L. (2016). Keratinases: Emerging trends in production and applications as novel multifunctional biocatalysts. Kuwait Journal of Science. 43. 118-127.

Kim, J. D. (2007). Purification and characterization of a keratinase from a feather degrading fungus Aspergillus flavus strain K-03. Microbiology 35:219-225.

Kumar,S., Strecher, G & Tamuru, K. (2016): MEGA7 Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 331870-1874.

Lateef, A., Adelere I.A & Gueguim–Kana, E.B. (2014). Bacillus safensis LAU 13: a novel source of Keratinase and its multi-functional biotechnically applications. Biotechnology & Biotechnological equipment 29, 54-63.

Lineweaver, H., & Burk, D. (1934). The Determination of enzyme constants. Journal of the American Chemical Society 56 (3), 658- 666

Ma, B., Qiao, X., Hou, X. & Yang, Y. (2016). Pure keratin membrane and fibers from chicken feather, Int. J. Biol. Macromol. 89, 614–621.

Minghai H., Wei L., Qiuya G., & Xiaobin, Y. (2012). Isolation and characterization of a keratinolytic protease from a feather-degrading bacterium Pseudomonas aeruginosa C11. African Journal of Microbiology Research, 6 (9): 2211-2221

Nnolim N. E., Okoh A. I., Nwodo U. U. (2020). Bacillus sp. fpf-1 produced keratinase with high potential for chicken feather degradation.Molecules 25,150. doi: https/doi/10.3390/molecules250715055

Nwadiaro, P., Ogbonna, A., Wuyep, P., & Adekojo, D. (2015). Keratinolytic Activity of Cladosporium and Trichoderma Species Isolated From Barbers’ Landfill. International Journal of Biosciences.6, 10:104-115.

Pawar, Vishakha., Prajapati, Anil., Akhani, Rekha., Patel, Darshan., & Pereira, J.Q., Lopes, F.C., Petry, M.V., da Costa Medina, L.F., & Brandelli, A (2014). Isolation of three novel Antarctic psychrotolerant feather degrading bacteria and partial purification of keratinolytic enzyme from Lysobacter sp. A03. Int. Biodeterior. Biodegrad. 88, 1–7.

Purchase, D.J. (2016). Microbial Keratinizes: characteristics biotechnological applications and potential. Handbook of microbial resources, chapter 10, Pp. 634-674.

Reichi, S., Borelli, M., & Geerling, G. (2011). Keratin films for ocular surface reconstruction. Biomaterials, 32: 3375-3386

Riffel, A & Brandelli, (2006): A Keratinolytic bacteria isolated from feather waste. Braz. J. microbiol. 37: 395-399

Sahni, N., Sahota, P., & Phutela, U. (2015). Bacterial keratinases and their prospective applications: A review. International Journal of Current Microbiology and Applied Sciences. 4. 768-783.

Sapna Rai., Ashish Sarah., & Kahita Sharma. (2014). Study of Keratin Degradation by Some Potential Fungal Isolates from Soil, Indian journal of applied research, Vol. 4: 8 ISSN 2249-555X

Selvan, K.K., & Vishnupriya, B. (2012). Biochemical and molecular characterization of microbial keratinase and its remarkable application. Int.J.Pham.Biol. Arch. 3:267-75.

Silveira, S., Jaeger, M., & Brandelli, A. (2009). Kinetic data and substrate specificity of a keratinase from Chryseobacterium sp. strain kr6. Journal of Chemical Technology and Biotechnology. 84. 361 - 366. doi: 10.1002/jctb.2048.

Silveira, S., Gemelli, S., Gemelli, S., Segalin, J., & Brandelli, A. (2012). Immobilization of keratinolytic metalloprotease from Chryseobacterium sp. strain kr6 on glutaraldehyde- activated chitosan. J. Microbiol Biotechnol 22: 818-825. doi:10.4014/jmb.1111.11048.

Tamura, K., & Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Boil. evol. 10: 512-526.

Tanabe, T., Okitsu, N., & Yamauchi, K. (2004). Fabrication and characterization of chemically crosslinked keratin films. Materials Science and Engineering: C. 24. 441-446. https://doi.org/10.1016/j.msec.2003.11.

Thys, F.S., Lucas, A., Riffel, P., Heeb, A., & Brandelli, A. (2004). Characterization of a protease of a feather – degrading micro bacterium species. letters in Applied microbiology.39:2. https://doi.org/10.1111/J.1472-765X.2004.01558.X.

United States Department of Agriculture (USDA). (2020). Livestock and poultry: World market and trade.

Wang, L., Liu, J., Li, X., Shi, J., Hu, J., Cui, R., Zhang, Z.L., Pang, D.W. & Chen, Y. (2011). Growth propagation of yeast in linear arrays of microfluidic chambers over many generations. Biomicrofluidics 5(4): 44118-441189.

Wang, B., Yang, W., McKittrick, J., & Meyers, M.A. (2015). Keratin: Structure, mechanical properties, occurrence in biological organisms, and efforts at bioinspiration. Progress in Materials Science. 3: 76:229–318.

Weber, K., & Osborn, M. (1975). The proteins -Academic Press New York

Yones, A & Metwalli, A. (2016). Effects of fish meal substitution with poultry by-product meal on growth performance, nutrients utilization and blood contents of juvenile nile tilapia (Oreochromis niloticus). J. Aquacul. Res. Dev. 7, 1-7