摘要:Background: Puffer fish skin tannery is an alternative to substitute the production of hide and animal skin in Indonesia that has been decreasing. To improve the quality of puffer fish leather, keratinase was needed by removing the thorns. Objective: The aim of this study was to screen bacteria that show keratinolytic activity. Methodology: The isolated bacterial strains were screened for the production of extracellular keratinase using skim milk agar. After incubation, the formations of a clear zone around the bacterial growth were observed. They were identified based on morphological enzyme activity and molecular identification. Bacterial screening and identification were conducted with descriptive method. The microbial activity tests were analyzed using a completely randomized design. Results: The result of the screening was that 3 of 5 kinds of strains exhibited caseinolytic activity (by showing the clear zone). Three Bacillus bacteria that were newly isolated from puffer fish waste-using a feather enrichment technique-were identified on the basis of 16S ribosomal RNA gene sequence analysis, physiological and carbohydrates assimilation tests. They were revealed as the strains of Bacillus thuringiensis for BRAW_PT isolate, Bacillus aerius for BRAW_PB isolate and Bacillus firmus for BRAW_PI isolate . The results of proteolytic enzymes assay showed that Bacillus firmus BRAW_PI has the highest protease and keratinase activity, which was 37.52±0.96 and 6.781±0.479 U mg1 consecutively. Conclusion: All bacteria obtained were the superior bacteria that can be used for the removal of thorns from puffer fish skin in the tanning process.