Wang Nan, Wang Kevin Yueju, Li GangQiang, Guo WenFang, Liu DeHu
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Department of Natural Sciences, Northeastern State University, Broken Arrow, OK 74014 USA.
Protein Expr Purif. 2015 Jul;111:75-81. doi: 10.1016/j.pep.2015.03.012. Epub 2015 Mar 31.
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.
凝乳酶能高效地使牛奶凝固，因此在商业奶酪生产中被广泛应用。传统的凝乳酶生产需要宰杀大量未断奶的小牛。在本研究中，合成了全长骆驼凝乳酶原基因，并将其克隆到pPIC9K载体中，然后将该载体插入酵母菌株巴斯德毕赤酵母GS115中。凝乳酶基因在酵母中的表达受AOX1诱导型启动子的控制。在实验室条件下，酵母系统产生了约37mg/L的重组酶。未处理的上清液的SDS-PAGE显示有两条分子量带，大小约为42kDa和45kDa。用N-糖苷酶F（PNGase F）处理上清液后，45kDa的条带消失，表明重组蛋白部分糖基化。在低pH条件下，重组凝乳酶原转化为成熟且有活性的凝乳酶。活性凝乳酶能够特异性地水解κ-酪蛋白。牛奶凝固活性的最适pH为5.04，温度范围为45-50°C。加入20-40mM CaCl2时检测到最大牛奶凝固活性。重组酶在20°C下、pH范围较宽（2.5至6.5）时8小时内具有高活性且稳定。还分析了重组酶的热稳定性。使用5L发酵罐实现了中试规模生产（300mg/L）。我们证明了在巴斯德毕赤酵母中表达骆驼凝乳酶基因可成为生产活性骆驼凝乳酶的优良系统，有望用于奶酪的商业生产。
