Expression and characterization of camel chymosin in Pichia pastoris

•We expressed a full-length camel prochymosin gene in transgenic yeast strain, Pichia pastoris.•Activated recombinant camel chymosin exhibited excellent milk-clotting activity.•Several factors critical to the milk-clotting activity of camel chymosin were investigated; including: optimal temperature...

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Bibliographic Details
Published in:Protein expression and purification 2015-07, Vol.111, p.75-81
Main Authors: Wang, Nan, Wang, Kevin Yueju, Li, GangQiang, Guo, WenFang, Liu, DeHu
Format: Article
Language:English
Subjects:
Animals
Camel chymosin
Camelus
Chymosin - biosynthesis
Chymosin - chemistry
Chymosin - genetics
Gene Expression
Milk-clotting
P. pastoris
Pichia - genetics
Pichia - metabolism
Recombinant
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Temperature
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Summary:•We expressed a full-length camel prochymosin gene in transgenic yeast strain, Pichia pastoris.•Activated recombinant camel chymosin exhibited excellent milk-clotting activity.•Several factors critical to the milk-clotting activity of camel chymosin were investigated; including: optimal temperature and thermostability, optimal pH and the effect of calcium ion concentrations. Chymosin efficiently coagulates milk and so is widely used in commercial cheese production. Traditional chymosin production requires the slaughter of a large numbers of unweaned calves. In the present study, a full-length camel prochymosin gene was synthesized and cloned into the pPIC9K vector, which was then inserted into the yeast strain, Pichia pastoris GS115. Expression of the chymosin gene in yeast was under the control of an AOX1 inducible promoter. The yeast system produced approximately 37mg/L of recombinant enzyme under lab conditions. SDS–PAGE of the raw supernatant revealed two molecular bands, which were approximately 42kDa and 45kDa in size. The 45kDa band disappeared after treatment of the supernatant with N-glycosidase F (PNGase F), indicating that the recombinant protein was partially glycosylated. When subjected to a low pH, recombinant prochymosin was converted into mature and active chymosin. The active chymosin was capable of specifically hydrolyzing κ-casein. A pH of 5.04, and temperature range of 45–50°C, was optimum for milk clotting activity. Maximum milk clotting activity was detected with the inclusion of 20–40mM CaCl2. The recombinant enzyme was highly active and stable over a wide pH range (from 2.5 to 6.5) at 20°C for 8h. Thermostability of the recombinant enzyme was also analyzed. Pilot-scale production (300mg/L) was attained using a 5L fermenter. We demonstrated that expression of the camel chymosin gene in P. pastoris could represent an excellent system for producing active camel chymosin for potential use in the commercial production of cheese.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2015.03.012