Constitutive expression, purification and characterization of bovine prochymosin in Pichia pastoris GS115.
Chymosin can specifically break down the Phe105-Met106 peptide bond of milk κ-casein to form insoluble para-κ-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B was chosen and constitutively expressed to a high level in Pichia pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of 24.2% was obtained for the purified enzyme, which appeared as a single band in SDS-PAGE having a molecular mass of approximate 36 kDa. Proteolysis assay showed that it specifically hydrolyzed κ-casein. It was stable at 25-50°C and had optimal activity at 37°C and pH 4.0. The activity of the recombinant chymosin was activated by cations such as Mn(2+), Fe(3+), Mg(2+) and Na(+), but inhibited by K(+), Co(2+), Zn(2+), Ni(2+), and to a lesser extent by Cu(2+). These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe and efficient enzyme suitable for use in cheese production.
Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China.
X P
XP
Jiang
M L
ML
Yin
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P
Chen
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Yang
D000818:Animals; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D012085:Chymosin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000378:metabolism; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008670:Metals / Q000378:metabolism; D008892:Milk / Q000378:metabolism; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature
D002364:Caseins; D020536:Enzyme Activators; D004792:Enzyme Precursors; D008670:Metals; D011994:Recombinant Proteins; C032443:prorennin; D012085:Chymosin