重组羧肽酶原B突变体C383A的纯化与性质研究.pdfVIP

308 食品与药品 Food and Drug 2010年第 12卷第09期 重组羧肽酶原B突变体C383A 的纯化与性质研究 张洛盛，吴 珊，吴 倩，李素霞* （华东理工大学生物反应器工程国家重点实验室，上海 200237 ） 摘 要：目的 纯化羧肽酶原B突变体C383A并研究其酶学性质。方法 用DEAE-FF离子交换树脂纯化C383A ，测定其动力 学参数，温度对酶活性的影响、温度稳定性、最适pH 以及pH稳定性等酶学性质，并与野生型比较。结果 纯化后获得高纯 度突变体C383A ，与野生型相比，催化效率及稳定性均有提高，但与底物的亲和力降低，随着温度的上升催化速率的增长 率低于野生型。结论 将重组羧肽酶原B第383位Cys突变为Ala可对酶的性质产生影响，与野生型比较，C383A提高了酶的 催化效率及温度和pH稳定性。 关键词：重组羧肽酶原B ；定点突变；纯化；酶学性质 中图分类号：Q555 文献标识码：A 文章编号：1672-979X(2010)09-0308-05 Study on Purification and Property of Recombinant Procarboxypeptidase B (Mutant C383A) Zhang Luo-sheng, WU Shan, WU Qian, LI Su-xia (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China) Abstract: Objective To study the purification and enzymatic property of procarboxypeptidase B (mutant C383A). Methods The mutant C383A was purified by DEAE-FF Ion-exchange Chromatography. The kinetic parameters and enzymatic property of mutant C383A which were compared with that of wild-type procarboxypeptidase B were measured, including the effects of temperature on activity and stability of mutant C383A, the optimal pH and the stability of pH. Results Compared with the wild-type procarboxypeptidase B, the purified mutant C383A with a high purity had the enhanced catalytic efficiency and stability. However, the affinity between mutant C383A and substrate was decreased and the increase of catalytic rate was lower than that of the wild-type as the temperature rose. Conclusion Cys383 mutation to Ala can affect the property of procarboxypeptidase B. Compared with the wild-type, the mutant C383A has the enhanced catalytic efficiency and stability to temperature and pH. Key Words: recombinant procarboxypeptidase