Carlsberg subtilisin from Bacillus licheniformis PB1 was investigated as a potential feed supplement, through immobilizing on bentonite for improving the growth rate of broilers. Initially, the pre-optimized and partially-purified protease was extracted and characterized using SDS-PAGE with MW 27.0 KDa. The MALDI-TOF-MS/MS spectrum confirmed a tryptic peptide peak with m/z 1108.496 referring to the Carlsberg subtilisin as a protein-digesting enzyme with alkaline nature. The highest free enzyme activity (30 U/mg) was observed at 50°C, 1 M potassium phosphate, and pH 8.0. the enhanced stability was observed when the enzyme was adsorbed to an inert solid support with 86.39 ± 4.36% activity retention under 20 optimized conditions. Additionally, the dried immobilized enzyme exhibited only a 5% activity loss after two-week storage at room temperature. Structural modeling (Docking) revealed that hydrophobic interactions between bentonite and amino acids surrounding the catalytic triad keep the enzyme structure intact upon drying at RT. The prominent hygroscopic nature of bentonite facilitated protein structure retention upon drying. During a 46-days study, supplementation of boilers' feed with the subtilisin-bentonite complex promoted significant weight gain i.e. 15.03% in contrast to positive control (p = 0.001).
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