Production, purification and characterization of keratinase from keratinase producing bacteria isolated from selected chicken Feature dumpsites in Ile Ife Nigeria
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Date
2021
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Department of Microbiology, Faculty of science, Obafemi Awolowo Universty, Ile Ife
Abstract
This research study focused on the isolation and identification of keratinase-producing bacteria from soil samples of selected chicken feather dump sites. It also explored the optimum conditions required for the production of keratinase by the isolated keratinase producing bacterium, thereby leading to the partial purification and characterization of the keratinase produced using its physicochemical and kinetic parameters. Soil samples were obtained from selected feather dumpsites in Ile-Ife from where keratinase producing bacteria isolated and purified using nutrient agar. The isolates were screened on skimmed milk agar and the isolates with high yield were subjected to submerged fermentation in order to select the best bacterial isolate for keratinase production. After 72 hours of incubation at a temperature of 37 oC, the mineral salt media were centrifuged at 4000 rpm for 20 minutes in a cold condition and the supernatants obtained were used as crude enzymes which were later quantified using keratinase assay and protein concentration. The optimum conditions of various parameters were investigated for the optimal production of keratinase by the isolate. Afterwards, all the optimum parameters were combined and used for the bulk production of crude enzyme. The crude enzyme produced was partially purified using 90 % Ammonium sulphate saturation and ion exchange chromatography and the partially purified enzyme was later characterized. A total of 110 bacteria were isolated and screened for keratinase production, 98 of the bacterial isolates tested positive to the screening process using Skimmed Milk agar. The best keratinase producer using the results of the keratinase assay and protein concentration from the submerged fermentation process was molecularly identified as Bacillus subtilis 2C-9B. The optimal temperature required for the production of keratinase by Bacillus subtilis 2C-9B was discovered to be 35 oC and at a pH 9.0. The carbon and nitrogen source that gave an optimal production of keratinase was discovered to be feather meal and yeast extract respectively. The bulk enzyme produced was partially purified using 90 % ammonium sulphate saturation and the result gave a 0.68-fold purification and this was further purified by ion exchange chromatography yielding an active protein peak at 4.9-fold purification. The purification process was followed by the characterization of the partially purified keratinase. An optimum pH in the alkaline region of pH 11.0 and optimum temperature of 45ÂșC was recorded. Metal ions like Ba2+, Hg2+ and Mn2+ inhibited the enzyme activity whereas Cu2+ and Pb2+ exhibited a slight inhibition. Phenylmethylsulphonyl fluoride (PMSF) completely inhibited the activity of the partially purified keratinase. However, EDTA did not inhibit the enzyme which implies that the partially purified enzyme belongs to the serine protease family and not the metalloprotease family. The study concluded that the keratinase produced by the isolated Bacillus subtilis 2C-9B strain was an alkalophilic enzyme which implies that it is suitable for industrial applications in leather processing and detergent industry.
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xvi, 150p
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Citation
AYEGUN,O.S (2021)production, purification and characterization of keratinase from keratinase producing bacteria isolated from selected chicken Feature dumpsites in Ile Ife Nigeria, Department of microbiology, Faculty of science, Obafemi Awolowo Universty ,Ile Ife