Isolation, Screening, Characterization and Identification of Alkaline Protease Producing Bacteria from Leather Industry Effluent

Abstract 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.