Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris
Akishev Z. Kiribayeva A. Mussakhmetov A. Baltin K. Ramankulov Y. Khassenov B.
May 2021Elsevier Ltd
Heliyon
2021#7Issue 5
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 cows milk and pH 4.0 for mares milk. The range 45–50 °C and 70 °C for cows and mares 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 mares 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.
Camel chymosin , Constitutive expression , Glycosylation , Milk coagulation , Protein purification , Recombinant DNA , Rennet
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National Center for Biotechnology, 13/5 Kurgalzhynskoye Road, Nur-Sultan, 010000, Kazakhstan
L.N.Gumilyov Eurasian National University, 2 Kanysh Satpayev Street, Nur-Sultan, 010008, Kazakhstan
National Center for Biotechnology
L.N.Gumilyov Eurasian National University
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