Recombinant chymosin of Camelus dromedarius in Pichia pastoris expression system: Purification and Enzymatic Profile

Natural chymosin production is an expensive and complex process associated with ethical issues. The article introduces recombinant chymosin Camelus dromedarius (rChn-Cam) isolated from a P. pastoris expression system and optimized for different nutrient media at different zeocin concentrations. The sequence of prochymosin gene was obtained from NCBI BLAST. GS115/his4 P. prastoris served as a producer strain. The pPICZ(alpha) B vector with the AOXI promoter made it possible to construct the expression cassette. The experiment involved methods of genetical engineering and strain cultivation. The recombinant His-Tag-labelled proteins were isolated by the method of metal-affinity chromatography and analyzed using PAG electrophoresis and Western-blot analysis. The molecular weight was determined by MALDI-TOF MS while the concentration was defined spectrophotometrically. The shuttle expression plasmid pPICZ(alpha)B/proCYM_camel_pp_IDT revealed that the cell mass expansion of P. pastoris GS115/his4 (Mut+) should be performed with a preliminary introduction of 0.5% methanol. After the transformation of P. pastoris GS115/his4 and obtaining a strain-producer of P. pastoris/pPICZ(alpha)B/proCYM_camel_pp_IDT, the rate of cell mass gain started to correlate with the zeocin concentrations in two different media. Medium YPD was not fornified and contained 50, 100, and 200 μg/mL zeocin. MediumYPD was fortified with 0.00004% biotin and 1% glycerol and included 50, 100, and 200 μg/mL zeocin. The strain-producer grew better in the medium with a zeocin concentration of 50 μg/mL. The mass of rChn-Cam was 35.673 kDa after isolation and purification. When the pH of the substrate rose from 5.0 to 6.5, the coagulation activity decreased by 24%. The thermal inactivation threshold of rChn-Cam was 40–45°C. The unit of coagulation activity decreased as the zeocin concentration went up. The rChn-Cam concentration was in inverse correlation with the substrate coagulation time. In this research, the rChn-Cam obtained in the expression system of P. prastoris proved to be a good alternative to rChn used in the cheese industry.