Ulyanovsk, Russian Federation
Ulyanovsk, Russian Federation
Ulyanovsk, Russian Federation
Ulyanovsk, Russian Federation
Ulyanovsk, Russian Federation
Ulyanovsk, Russian Federation
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.
genetic construct, recombinant chymosin, Camelus dromedarius, vector, plasmid, yeast Pichia pastoris, milk-setting activity, thermostability, chymosin purification
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