Journal
BRAZILIAN JOURNAL OF MICROBIOLOGY
Volume 54, Issue 2, Pages 739-752Publisher
SPRINGER
DOI: 10.1007/s42770-023-00992-6
Keywords
Cysteine protease; Skimmed milk agar; Metal ions; Protease inhibitors; MALDI-TOF; Protease industrial applications
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Bacterial proteases were isolated from skimmed milk agar media through serial dilution. The strains were identified as Bacillus subtilis and designated as A1 (MT903972), A2 (MT903996), A4 (MT904091), and A5 (MT904796) based on microbial biomass production, biochemical tests, protease-specific activity, and 16s RNA gene sequencing. Among them, A4 Bacillus subtilis exhibited the highest protease-specific activity of 76,153.84 U/mg. The identified cysteine protease showed great potential in various industrial applications.
Bacterial proteases have extensive applications in various fields of industrial microbiology. In this study, protease-producing organisms were screened on skimmed milk agar media using serial dilution. Through microbial biomass production, biochemical tests, protease-specific activity, and 16 s RNA gene sequencing, the isolates were identified as Bacillus subtilis and submitted to NCBI. The strain accession numbers were designated as A1 (MT903972), A2 (MT903996), A4 (MT904091), and A5 (MT904796). The strain A4 Bacillus subtilis showed highest protease-specific activity as 76,153.84 U/mg. A4 Bacillus subtilis was unaffected by Ca2+, Cu2+, Fe2+, Hg2+, Mg2+, Na, Fe2+, and Zn2+ but was inhibited by 80% by Mn2+ (5 mM). The protease activity was inhibited by up to 30% by iodoacetamide (5 mM). These findings confirm the enzyme to be a cysteine protease which was further confirmed by MALDI-TOF. The identified protease showed 71% sequence similarity with Bacillus subtilis cysteine protease. The crude cysteine protease significantly aided in fabric stain removal when added to a generic detergent. It also aided in the recovery of silver from used X-ray films and de-hairing of goat skin hides and showed decent application in meat tenderization. Thus, the isolated cysteine protease has high potential for industrial applications.
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