Expression and characterization of camel chymosin in Pichia pastoris

文献类型： 外文期刊

第一作者： Wang, Nan

作者： Wang, Nan;Li, GangQiang;Guo, WenFang;Liu, DeHu;Wang, Kevin Yueju

作者机构：

关键词： Camel chymosin;P. pastoris;Milk-clotting;Recombinant;pH;Temperature

期刊名称：PROTEIN EXPRESSION AND PURIFICATION （ 影响因子：1.65； 五年影响因子：1.548 ）

ISSN：

年卷期：

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收录情况： SCI

摘要： 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 37 mg/L of recombinant enzyme under lab conditions. SDS-PAGE of the raw supernatant revealed two molecular bands, which were approximately 42 kDa and 45 kDa in size. The 45 kDa 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 kappa-casein. A pH of 5.04, and temperature range of 45-50 degrees C, was optimum for milk clotting activity. Maximum milk clotting activity was detected with the inclusion of 20-40 mM CaCl2. The recombinant enzyme was highly active and stable over a wide pH range (from 2.5 to 6.5) at 20 degrees C for 8 h. Thermostability of the recombinant enzyme was also analyzed. Pilot-scale production (300 mg/L) was attained using a 5 L 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. (C) 2015 Elsevier Inc. All rights reserved.

分类号： Q51

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