Background A wide variety of bacterial species produces protease enzyme, and the application of the same enzyme has been manipulated precisely and used in various biotechnological areas including industrial and environmental sectors. The main aim of this research study was to isolate, screen, and identify alkaline protease-producing bacteria that were sampled from leather industry effluent present in the outer skirts of Addis Ababa, Ethiopia. Purpose To isolate and characterize the alkaline protease-producing bacteria from leather industrial effluents. Methods Samples are collected from Modji leather industrial effluents and stored in the microbiology lab. After isolated bacteria from effluent using serial dilution and followed by isolated protease-producing bacteria using skim milk agar media. After studying primary and secondary screening using zonal inhibition methods to select potential protease-producing bacteria using skim milk agar media. Finally, to identify the potential bacteria using biochemical methods, bacterial biomass, protease activity, and gene sequencing (16S rRNA) method to finalize the best alkaline protease producing bacteria identified. Results First twenty-eight different bacterial colonies were isolated initially from the leather industry effluent sample situated at the Modjo town of Ethiopia. The isolated bacteria were screened using the primary and secondary screening method with skim milk agar medium. At the primary level, we selected three isolates namely ML5(14 mm), ML12(18 mm), and MS12 (15 mm), showing the highest zone of proteolysis as a result of casein degradation on the agar plates were selected and subjected to primary screening. Further secondary screening confirmed that the zone of inhibition methods ML5 (14.00±0.75 mm), ML12 (19.50±0.66 mm), and MS12 (15.00±1.32 mm) has efficient proteolytic activity and can be considered as effective protease producer. The three isolates were then subjected to morphological and biochemical tests to identify probably bacterial species, and all the three bacterial isolates were found out to be of Bacillus species. The shake flask method was carried out to identify the most potent one having greater biomass production capabilities and protease activity. ML12 isolated from leather effluent waste showed the highest protease activity (19 U/ml), high biomass production, and the same was subjected to molecular identification using 16s sequencing and a phylogenetic tree was constructed to identify the closest neighbor. The isolate ML12 (Bacillus cereus strain -MN629232.1) is 97.87% homologous to Bacillus cereus strain (KY995152.1) and 97.86% homologous to Bacillus cereus strain (MK968813.1). Conclusions This study has exposed that from twenty-eight different bacterial samples isolated from leather industry effluent; further primary and secondary screening methods were selected three potential alkaline protease strains. Finally, based on its biochemical identification, biomass, and protease activity, ML12 (Bacillus cereus strains) is the best strain identified. The alkaline protease has the significant feature of housing potent bacterial species for producing protease of commercial value.

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BackgroundA wide variety of Bacterial species produces protease enzyme and the application of same enzyme have been manipulated precisely and used in various biotechnological areas including industrial and environmental sectors. The main aim of this research study was to isolate, screen and identify protease producing bacteria which were sampled from leather industry effluent present in the outer skirts of Addis Ababa, Ethiopia.PurposeTo isolated alkaline protease producing bacteria from leather industrial effluents and to characterization (Secreening and identification).MethodsSample collected from Modji leather industrial effluents and stored in the microbiology lab. After isolated bacteria from effluent using serial dilution and followed by isolate protease producing bacteria using skim milk agar media. After studying Primary and secondary screening using zonal inhibition methods to select potential protease producing bacteria using skim milk agar media. Finally to characterization and identification of potential bacteria using biochemical methods, protein estimation, biomass, protease assay and gene sequencing (16S rRNA) method to finalized best protease producing bacteria. ResultsTwenty-eight different bacterial colonies were isolated initially from the leather industry effluent sample situated at Modjo town of Ethiopia. The isolated bacteria were screened using primary screening method with skim milk agar medium. Three isolates namely MS12, ML5 and ML12 showing highest zone of proteolysis as a result of casein degradation on the agar plates were selected and subjected to secondary screening. Further secondary secreening confirmed that MS12, ML5 and ML12 has efficient proteolytic activity and can be considered as potent protease producer. The three isolates were then subjected to morphological and biochemical tests to identify probably bacterial species and all the three bacterial isolates were found out to be of Bacillus species. Shake flask method was carried out to identify the most potent one having greater biomass production capabilities, protein quantity and protease activity. ML12 isolated from leather effluent waste showed highest Protein(170mg/ml), Protease activity(19U/ml), high biomass production and the same was subjected to molecular identification using 16s sequencing and a Phylogenetic tree was constructed to identify the closest neighbor. The isolate ML12 is 97.87% homologous to Bacillus cereus strain (KY995152.1) and 97.86% homologous to Bacillus cereus strain (MK968813.1).ConclusionsThis study has revealed that the leather industry effluent site has significant feature of housing potent bacterial species producing protease of commercial value. Being one among the most widely used enzyme, comparatively. Protease holds a larger scope for research and commercialization any other type of enzymes. There is a need to develop novel protease enzymes for further necessary applications of these enzymes. Moreover, enzyme produced by bacteria which are present in effluents are a greater boon to establish the significance of converting industrial wastes to a highly valuable enzymes especially like proteases.

Purpose: To investigate the detailed analysis of alkaline protease production, media optimization, protease assay, mass production, and application in feather degradation by isolates of Bacillus cereus strain isolated from leather industry effluent, Ethiopia.Methods: In this study, media optimization was subjected to nine different parameters like fermentation time, temperature, pH, Substrate concentration, carbon sources, nitrogen sources, metal ions, Inoculum size, and sodium chloride concentration to check the maximum alkaline protease production using Bacillus cereus. Ammonium sulfate and dialysis were used to partially purify the enzyme from Bacillus cereus. SDS-PAGE was used to test the activity and total protein content of the partially purified enzymes, and then extracellular alkaline protease from Bacillus cereus was used to examine hydrolyzed chicken feathers, and the findings were reported.Results: The maximum enzyme activity of crude alkaline protease from Bacillus cereus was achieved at optimized conditions. The specific enzyme activity of partially purified alkaline protease after dialysis (123.35U/mg) showed high activity compared to the crude protease (24.45U/mg). Finally, the partially purified enzyme was tested for its potential to degrade feather waste. Partially purified enzyme demonstrated significant feather degradation of 76.5% on the fifth day at optimized temperature and enzyme concentration.Conclusions: Bacillus cereus was found to cause considerable feather deterioration in this investigation. It's was also been discovered that enzyme activity gets enhanced at a specific optimized condition. Compared to the crude enzyme, partially purified and dialyzed enzymes showed considerable change in the enzyme activity, indicating that they have the potential to break down feathers.