Characterization of high-yield Bacillus subtilis cysteine protease for diverse industrial applications

Abstract

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.

Keywords: Cysteine protease; MALDI-TOF; Metal ions; Protease industrial applications; Protease inhibitors; Skimmed milk agar.

Fig. 1

Screening and identification of protease-producing bacterial strains (A1–A5) from soil. a Primary screening of protease-producing microorganisms on skimmed milk agar media. b Secondary screening of protease-producing organisms on nutrient agar plates by streak plate method to obtain isolated colonies. c Gram stained images of isolates. d Zone of hydrolysis of protease-producing bacterial isolates on skimmed milk agar plates showing zone of proteolysis

Fig. 2

16S rRNA gene sequencing and phylogenetic analysis. Phylogenetic tree constructed from 16 s rRNA gene sequencing of Bacillus subtilis using a maximum likelihood method

Fig. 3

Effect of pH and temperature on A4 cysteine protease enzyme. a Effect of protease activity at different pH ranging from pH 2 to pH 12. b Effect of protease activity at different temperatures ranging from 4 to 80 °C

Fig. 4

MALDI-TOF and peptide mass fingerprinting (PMF) identification of cysteine pProtease isolated from Bacillus subtilis. MASCOT search followed by PMF analysis showed the matched amino acid residues (in bold) of the peptide fragments with the putative cysteine protease of Bacillus subtilis

Fig. 5

Blood stain removal, detergent activity, and silver recovery from used X-ray films with B. subtilis cysteine protease. a Blood stain removal: sterile cotton cloth was stained with blood and dried. First blood stain – untreated (i, iv), second stain was washed with distilled water (ii, v), and the third blood stain was treated with 200–500 µl of Bacillus subtilis cysteine protease for 30 s (iii, vi). The first row of images represents before treatment (i, ii, iii), and the second row represents images post treatment with distilled water or protease. b Washing performance of protease from Bacillus subtilis in combination with commercial detergent. c Decomposition of gelatin layer of X-ray film by protease

Fig. 6

De-hairing of the goat skin and meat tenderization by B. subtilis cysteine protease. Enzymatic de-hairing of goat skin: a skin sample treated with skimmed milk broth (control), b skin sample treated with distilled water, c skin sample treated with protease enzyme. Meat tenderization: scanning electron micrographs (SEM) of cooked intra-muscular tissue of buffalo meat. d Untreated, e protease-treated sample, and f raw papaya extract treated sample. Scale bars are adjusted to 10 µm per unit