BackgroundCalf rennet is considered the traditional source of milk clotting enzyme (MCE). However, increasing cheese consumption with decreasing the calf rennet supply had encouraged the quest for new rennet alternatives. The purpose of this study is to acquire more information about the catalytic and kinetic properties of partially purifiedBacillus subtilisMK775302 MCE and to assess the role of enzyme in cheese manufacture.ResultsB. subtilisMK775302 MCE was partially purified by 50% acetone precipitation with 5.6-fold purification. The optimum temperature and pH of the partially purified MCE were 70 °C and 5.0, respectively. The activation energy was calculated as 47.7 kJ/mol. The calculatedKmandVmaxvalues were 36 mg/ml and 833 U/ml, respectively. The enzyme retained full activity at NaCl concentration of 2%. Compared to the commercial calf rennet, the ultra-filtrated white soft cheese produced from the partially purifiedB. subtilisMK775302 MCE exhibited higher total acidity, higher volatile fatty acids, and improved sensorial properties.ConclusionsThe partially purified MCE obtained in this study is a promising milk coagulant that can replace calf rennet at a commercial scale to produce better-quality cheese with improved texture and flavor.Supplementary InformationThe online version contains supplementary material available at 10.1186/s43141-023-00513-w.