Milk‐Clotting Protease From Bacillus stercoris NCCP ‐3139: A Potential Microbial Rennet for Cheese Production

ABSTRACT Milk‐clotting enzymes (MCEs) are essential biocatalysts in cheese manufacture. However, limited availability and ethical concerns associated with calf rennet have intensified the search for microbial alternatives. This study reports the purification and characterization of a milk‐clotting protease from Bacillus stercoris NCCP‐3139, a soil isolate from Dera Ismail Khan, Pakistan. The strain was identified as B. stercoris by standard morphological and biochemical assays and 16S rRNA gene sequencing (98.75% similarity to B. stercoris JCM 30051). The enzyme was purified by ammonium sulfate precipitation, dialysis, DEAE–cellulose ion‐exchange chromatography and Sephacryl S‐200 gel filtration chromatography. Milk‐clotting activity was assessed in goat, sheep, cow, buffalo and camel milk. The isolate exhibited pronounced casein hydrolysis and significant milk‐clotting activity ( p < 0.05), consistent with the production of a rennin‐like protease. Sheep and buffalo milk showed superior clotting performance compared with other species ( p < 0.05). Purification increased specific activity from 1.6 to 5.0 U/mg and the milk‐clotting‐to‐proteolytic activity (PA) ratio from 50 to 550, indicating significant catalytic selectivity. The enzyme displayed optimal activity at 55°C and pH 9.0, and was activated by Mn 2+ and Sr 2+ but strongly inhibited by EDTA, Hg 2+ and Zn 2+ . SDS–PAGE revealed a single band, consistent with a monomeric enzyme. Thus isolated B. stercoris NCCP‐3139 produces protease with significant specificity and selectivity indicating its potential as a microbial alternative to animal rennet in cheese production.