CONSTRUCTION OF BOVINE CHYMOSIN-PRODUCING LACTOCOCCUS LACTIS STRAIN

Tessália Diniz Luerce Saraiva (UFMG); Kátia Morais Costa (UFMG); Marcela Pacheco de Azevedo (UFMG); Jucimar Zacaria (UCS); Ana Paula Longaray Delamare (UCS); Sergio Echeverrigaray (UCS); Vasco Ariston de Carvalho Azevedo (UFMG); Anderson Miyoshi (UFMG)

Chymosin, a proteolytic enzyme produced in the abomasum of suckling calves, is synthesized in vivo as proenzyme, the prochymosin. Then, under acidic pH, it is cleaved to the active form, chymosin. Chymosin efficiently converts liquid milk to a semisolid like cottage cheese, allowing it to be retained for longer periods in the stomach environment. Thus, due to its high capacity of clotting milk, it has been widely used on cheese industry. However, when this enzyme is extracted from the rennet stomach of newborn calves, it may contain up to 15% of pepsin, which can compromise cheese quality. Thus, in order to produce an enzyme with a higher grade of purity, we propose the production of chymosin in Lactococcus lactis, a lactic acid bacterium widely used as starter culture for the production of fermented dairy products. L. lactis is a good and safe alternative for the production of recombinant proteins of biotechnological interest due to its “GRAS” (Generally Regarded As Safe) status. Here, we describe the construction of a recombinant L. lactis strain expressing the secreted form (SEC) of the Pro-Chymosin B from Bos taurus based in a xylose-inducible expression system. The ORF of the Pro-Chymosin B was synthesized, based on the codon usage of L. lactis, and PCR-amplified. The amplicon, corresponding to the SEC form of the Pro-Chymosin B coding sequence, was cloned into the pCR®-Blunt II-TOPO®, subcloned into pXylT:SEC expression vector and then transferred to L. lactis. The production capacity of the L. lactis strain was determined by immunoblot assays. Pro-Chymosin B was efficiently produced in L. lactis, although it had been observed that the recombinant protein remained stacked in the cell envelope; in consequence, no clotting or proteolytic activity of the secreted form of the Pro-Chymosin B was observed. Therefore, cloning of the Bos taurus Pro-Chymosin B, in its active form (Chymosin B), may be a more interesting strategy for the attainment of this enzyme, since its lower molecular weight and folding could facilitate its targeting to the extracellular medium. In order to investigate this hypothesis, we constructed another L. lactis strain, now expressing the secreted form of the bovine Chymosin B. Further experiments using this new strain are now in progress and will allow definition of the potential of this strain as milk leaven and Chymosin producer.