Cloning and overproduction of chymosin from Bacillus spp in Escherichia coli origami

Making cheese is simply a process of turning a liquid (milk) in-to a solid. Rennet has an important role in this process which is a complex set of enzymes where chymosin is the key component. The aim of this study was identifying the Bacillus genus with the ability of producing the enzyme. 50/75 isolates from soil of Gandom Beryan area of Kerman, Iran, determined as bacillus spp via classical and PCR methods. The presence and over production of the cltA gene (encoding chymosin) were investigated by performing cloning in Escherichia coli (E. coli) origami and functional cloning confirmed by using real-time reverse transcription – polymerase reaction (Real-time PCR) and clonal selection. The results showed functional cloning of the gene in Escherichia coli origami. Among 50 isolates of Bacillus spp, 4 isolates (8%) showed the capacity to produce the gene. Phylogenetic characterization analysis of 16S rRNA gene of the Bacillus spp determined the strains as Bacillus cereus. According to the results of this study, expression of the gene via Real-time PCR showed their ability to produce clt A gene and rennet. Although to further confirmation, a larger samples needs to be performed and additional tests are required. Due to animal rennet deficiency, determining such accessible and cheaper sources in producing rennet is important.