The Development of Biotechnological Techniques for the Processing and Use of Bovine Chymosin on an Industrial Scale

Abstract

Chymosin is an enzyme, traditionally obtained from the fourth stomach of young ruminants, used in the coagulation of milk to make cheese. In the present work we will address other aspects that are difficult to resolve during the processing of this protein, its activation, purification process and alternatively, its immobilization in various chromatographic matrices that allow its manipulation under more technically industrial and economically beneficial industrial conditions. The process consisted of cloning the coding gene for this enzyme, using optimized codons for its overexpression in Escherichia coli, with the pBAD/HIS vector under the control of the pBAD promoter inducible by L-arabinose, as it is a more economically profitable process. The process consisted of cloning the coding gene for this enzyme, using optimized codons for its overexpression in Escherichia coli, with the pBAD/HIS vector under the control of the pBAD promoter inducible by L-arabinose, as it is a more economically profitable process. Overexpression of the gene generates inclusion bodies that can be denaturalized by washing with NaOH, for subsequent opening of the protein by dilution and adjustment of the pH with glycine, in a more easily manipulated way. Once unfolded, prochymosin is activated by a drastic change in pH, first at pH 2 and then rising to pH5 to increase its activation process. After this process, a highly active protein was obtained, tested under its natural substrate, milk. Due to the complexity of the activation-reactivation- purification process that takes place at very acidic pHs, its control becomes difficult, which is why new alternatives are being sought to overcome this inconvenience. The more conservative alternative found was the use of immobilization techniques on different polymeric supports (agarose based) and on magnetic particles (magnetic nanoparticles), previously functionalized, so that in a single step we could carry out the purification process and protein activation. When using polymeric supports, the immobilization results were very poor (below 20%), mainly due to pore clogging problems in this type of resin; whilst, the use of magnetic nanoparticles completely changed the scenario, obtaining yields of almost 100% and retention of enzymatic activity at values close to 85%.