Alkaline Protease Production from Bacillus gibsonii 6BS15-4 Using Dairy Effluent and Its Characterization as a Laundry Detergent Additive

Abstract

Protease is a widely used enzyme particularly in the detergent industry. In this research, we aimed to isolate alkaline protease-producing bacteria for characterization as a laundry detergent additive. The screening of alkaline protease production was investigated on basal medium agar plus 1% skim milk at pH 11, with incubation at 30°C. The highest alkaline protease-producing bacterium was 6BS15-4 strain, identified as Bacillus gibsonii by 16S rRNA gene sequencing. While the optimum pH was 12.0, the strain was stable at pH range 7.0-12.0 when incubated at 45°C for 60 min. The alkaline protease produced by B. gibsonii 6BS15-4 using dairy effluent was characterized. The optimum temperature was 60°C and the enzyme was stable at 55°C when incubated at pH 11.0 for 60 min. Metal ions K⁺, Mg²⁺, Cu²⁺, Na⁺, and Zn²⁺ exhibited a slightly stimulatory effect on enzyme activity. The enzyme retained over 80% of its activity in the presence of Ca²⁺, Ba²⁺, and Mn²⁺. Thiol reagent and ethylenediaminetetraacetic acid did not inhibit the enzyme activity, whereas phenylmethylsulfonyl fluoride significantly inhibited the protease activity. The alkaline protease from B. gibsonii 6BS15-4 demonstrated efficiency in blood stain removal and could therefore be used as a detergent additive, with potential for various other industrial applications.

Keywords: Alkaline protease; Bacillus gibsonii; dairy effluent; isolation; laundry detergent.

Fig. 1. Alkaline protease-producing bacteria B. gibsonii 6BS15-4 on BMSM agar plates showing zone of proteolysis when incubated at 30°C for 72 h (scale bar = 10 mm).

Fig. 2. Time course of cellular growth (-□-) and alkaline protease production (-△-) of B. gibsonii 6BS15-4 when cultured in BMSM, pH 11, and incubated at 30°C with shaking at 120 rpm for 192 h.

Fig. 3. The effects of various pH on the alkaline protease activity (-●-) and stability (-○-) of B. gibsonii 6BS15-4.

Fig. 4. The effects of various temperatures on the alkaline protease activity (-▲-) and stability (-△-) of B. gibsonii 6BS15-4.

Fig. 5. Application of alkaline protease of B. gibsonii 6BS15-4 on blood stain removal.

Fig. 6. Relative protein hydrolysis of blood (-■-), egg solution (-▲-), chicken feathers (-□-) and milk (-○-) by alkaline protease of B. gibsonii 6BS15-4 at 30°C for 3 days.