Isolation of lysozyme producing Bacillus subtilis Strains, identification of the new strain Bacillus subtilis BSN314 with the highest enzyme production capacity and optimization of culture conditions for maximum lysozyme production

Abstract

• Screening, isolation and characterization of lysozyme-producing Bacillus subtilis strains. • Bacillus subtilis BSN314 was found to have highest biomass production capacity. • Analysis of results was based on two techniques; single factor and orthogonal method. • By single factor, influence of medium composition and growth conditions was determined. Lysozyme is a naturally occurring enzyme that is ubiquitous and plays an important role in innate immunity through its potent bacteriolytic activity. The main objective of this study was to isolate, screen and identify lysozyme-producing Bacillus subtilis strains by 16S rDNA sequencing. Of the four Bacillus subtilis strains isolated (B18, B66, B70, and B124), only one (B66) was selected for further study because it exhibited the comparatively highest enzymatic activity. The 16S rDNA sequence of strain B66 was submitted to the Genbank database with accession number OM327451 and was assigned the name Bacillus subtilis BSN314. Bacillus subtilis BSN314 was selected for further study to maximize biomass production. To obtain the maximum biomass product of Bacillus subtilis BSN314, two statistical methods were applied, one related to the nutritional conditions affecting the growth medium (chemical components), called the single factor optimization method, and the other showing their effects on the cultivation conditions (orthogonal method). The results showed that the composition of the culture medium (carbon, nitrogen, inorganic salts and surfactants) and the culture conditions (temperature, pH, shaking speed and percentage of inoculum) have a specific influence on the biomass production of Bacillus subtilis BSN314. The culture medium containing the combination of A2B3C1D2 with 1.5% glucose, 2.5% soybean peptone, 0.5% K 2 HPO 4 , and 0.1% Tween-20 was determined to be the optimal medium for biomass production whereas, the order of culture media based on the effects of the selected conditions having lysozyme activity is as follows; glucose % > soyapeptone % > Tween-20 % > K 2 HPO 4 %. The results showed that the A2B3C1D2 ratio had the highest enzyme activity of 55 U/mL.