Optimization of alkaline protease production by a strain of Bacillus licheniformis

Abstract:

[Background] Alkaline protease, a fermentation product of many Bacillus species, is an important enzyme in industry. [Objective] An alkaline protease-producing strain was isolated by casein medium from an environmental sample. The passage number, carbon source, nitrogen source, metal ions, phosphate, initial pH, inoculum amount and temperature of the fermentation were optimized to increase the alkaline protease production of the strain and to reduce the cost of fermentation. [Methods] The isolates were identified via Gram staining, scanning electron microscopy, physiological and biochemical tests, and 16S rRNA sequence analysis. The fermentation conditions were optimized by single factor test, Plackett-Burman experiment, the steepest ascent method, and response surface methodology. The experimental data were analyzed in Minitab. [Results] The strain was identified as B. licheniformis and named B. licheniformis NWMCC0046. The optimized formula of the fermentation medium was determined as soybean meal 50.00 g/L, glucose 10.00 g/L, yeast extract 13.46 g/L, CaCl₂ 0.50 g/L, Na₂HPO₄·12H₂O 4.00 g/L, and KH₂PO₄ 0.30 g/L. The fermentation conditions were optimized as pH 7.5, 34.81 ℃ and the inoculum amount of 4.13%. Under these conditions, the alkaline protease activity reached 165.41 U/mL in 48 h of shake flask fermentation, which was 8.04 times higher than that before optimization. [Conclusion] The optimization of the medium formula and fermentation conditions improved the ability of B. licheniformis to produce alkaline protease, providing a reference for subsequent research on alkaline protease.

Key words:Bacillus licheniformis;alkaline protease;Plackett-Burman design;the steepest ascent method;response surface methodology