Abstract:
Camel chymosin can be efficiently employed to produce cheese. Traditionally the rennet enzyme produced by the
glands of the fourth stomach of ruminant animals (abomassum) is used in cheese making. Full-length Camelus
bactrianus (Bactrian camel) prochymosin gene was synthesized and constitutively expressed in Pichia pastoris cells
under glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter. It was purified by sequential anion and
cation exchange chromatography. SDS-PAGE analysis resulted in two bands, approximately 42 and 35 kDa. The
42 kDa band vanished when the sample was treated with endoglycosidase H, indicating that the recombinant
protein is partially glycosylated. Optimal pH for the activity of the highest-purity recombinant chymosin was pH
4.5 for cow's milk and pH 4.0 for mare's milk. The range 45–50 C and 70 C for cow's and mare's milk types,
respectively, was found to be the most appropriate for maximal relative milk-clotting activity. Concentration of
CaCl2 that ensured the stability of the chymosin milk-clotting activity was between 20 and 50 mM with an optimum at 30 mM. Milk-clotting activity of camel recombinant chymosin and ability to make curd was successfully
tested on fresh mare's milk. Pichia pastoris strain with integrated camel chymosin gene showed high productivity
of submerged fermentation in bioreactor with milk-clotting activity 1412 U/mL and 80 mg/L enzyme yield. These
results suggest that the constitutive expression of the camel chymosin Camelus bactrianus in the yeast Pichia
pastoris has good prospects for practical applications.
