前导肽区域N-糖基化影响骆驼凝乳酶原在毕赤酵母中的分泌表达和热稳定性研究  

N-glycosylation in the Propeptide Improved Thermostability of Camel Prochymosin and Enhanced Its Secretion in Pichia pastoris

作　　者：王楠[1] 杨彩峰 彭华康 郭文芳[1] 王梦琪 李刚强[1] 刘德虎[1] WANG Nan;YANG Caifeng;PENG Huakang;GUO Wenfang;WANG Mengqi;LI Gangqiang;LIU Dehu(Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]中国农业科学院生物技术研究所,北京100081

出　　处：《中国农业科技导报》2022年第10期71-78,共8页Journal of Agricultural Science and Technology

基　　金：国家自然科学基金项目(31901661)。

摘　　要：骆驼凝乳酶原在毕赤酵母中的表达量较低,不能满足产业化需求。为提高骆驼凝乳酶原的表达量,采用定点突变的方法,分别在其前导肽的第11、26、34、35、38、39位氨基酸处引入1个N-糖基化位点,构建成6种骆驼凝乳酶原的N-糖基化突变体:chy11、chy26、chy34、chy35、chy38和chy39,并分别导入到毕赤酵母中进行分泌表达。测定和比较了野生型(wild)和各突变体骆驼凝乳酶原的表达量。Chy34、chy35、chy38和chy39的表达量显著提高,其中chy34的表达量最高。6种突变体均保持前导肽自切割活性。在50℃保温8 h,wild酶活完全丧失,而突变体仍有20%~80%的相对酶活,表明6种突变体的热稳定性均显著增强。研究结果为提高骆驼凝乳酶原在毕赤酵母中的表达量和增强热稳定性提供了新的研究思路。The expression level of camel prochymosin in Pichia pastoris is low and cann’t meet the needs of industrialization. In order to improve the expression of camel prochymosin,N-glycosylation sites were introduced into the 11,26,34,35,38,39amino acids of propeptide region,respectively,to constructe 6 N-glycosylation mutants:chy11,chy26,chy34,chy35,chy38 and chy39. These mutants were introduced into P. pastoris for secretory expression and the expression levels of wild type and mutants were measured and compared. The secretory expression of chy34,chy35,chy38 and chy39 increased significantly,and the expression level of chy34 was the highest. The leading peptide of all 6 mutants maintained self cleavage activity. At 50 ℃ for 8 h,the enzyme activity of wild was completely lost,while the mutants still had 20%~80% relative enzyme activity,indicating that the thermotability of the 6 mutants was significantly enhanced. The results provided new research idea for improving the expression of camel prochymosin in P. pastoris and enhancing thermostability.

关 键 词：骆驼凝乳酶原 N-糖基化 毕赤酵母 分泌表达 热稳定性 

分 类 号：Q78[生物学—分子生物学]