AimsThis study was an attempt to create a novel milk clotting procedure using a recombinant bacterium capable of milk coagulation. Methods and ResultsThe Rhizomucor pusillus proteinase (RPP) gene was sub-cloned into a pALF expression vector. The recombinant pALF-RPP vector was then electro-transferred into Lactococcus lactis. Finally, the milk coagulation ability of recombinant L. lactis carrying a RPP gene was evaluated. Nucleotide sequencing of DNA insertion from the clone revealed that the RPP activity corresponded to an open reading frame consisting of 1218bp coding for a 4345kDa RPP protein. The RPP protein assay results indicated that the highest RPP enzyme expression with 870 Soxhlet units (SU) per ml and 7914 SU/OD were obtained for cultures which were incubated at pH 55 and 30 degrees C. Interestingly, milk coagulation was observed after 205 min of inoculating milk with recombinant L. lactis carrying the RPP gene. ConclusionThe recombinant L. lactis carrying RPP gene has the ability to function as a starter culture for acidifying and subsequently coagulating milk by producing RPP as a milk coagulant agent. Significance and Impact of the StudyCreating a recombinant starter culture bacterium that is able to coagulate milk. It is significant because the recombinant L. lactis has the ability to work as a starter culture and milk coagulation agent.