白假丝酵母环核苷酸磷酸二酯酶2的异源表达、纯化与酶学特性分析  

Heterologous Expression,Purification and Enzymatic Analysis of Candida albicans Cyclic Nucleotide Phosphodiesterase 2

作　　者：李赤霞 陈滢 张萌[1] 陈玉娟[1] 田元元[1] 王友升[1,3] LI Chixia;CHEN Ying;ZHANG Meng;CHEN Yujuan;TIAN Yuanyuan;WANG Yousheng(Beijing Advanced Innovation Center for Food Nutrition and Human Health,School of Light Industry,Beijing Technology&Business University(BTBU),Beijing 100048,China;Shandong KEEPFIT Biotech Co.Ltd.,Rizhao 276800,China;Rizhao HUAWEI Institute of Comprehensive Health Industries,Rizhao 276800,China)

机构地区：[1]北京食品营养与人类健康高精尖创新中心,北京工商大学轻工科学技术学院,北京100048 [2]山东凯普菲特生物科技有限公司,山东日照276800 [3]日照华伟大健康产业研究院,山东日照276800

出　　处：《食品科学》2020年第22期82-87,共6页Food Science

基　　金：国家自然科学基金面上项目(31972127);北京市教委科技计划重点项目(KZ201910011013)。

摘　　要：为获得白假丝酵母(Candida albicans)环核苷酸磷酸二酯酶2(phosphodiesterases 2,PDE2),以C.albicans基因组为模板,经聚合酶链式反应扩增获得目的基因,构建重组质粒pET28a-C.albicans pde2,转化至大肠杆菌BL21中,获得稳定表达的基因工程菌。所表达的蛋白通过亲和层析柱(Ni-NAT)、离子交换层析柱(Q-Sepharose)和分子筛层析柱(Sephacryl S200)逐步进行纯化。应用高效液相色谱方法测定纯化的PDE2蛋白(约62 kDa)对单底物及双底物的水解特性,证明PDE2在0.385 mg/mL质量浓度下,对0.4 mmol/L cAMP和cGMP的水解率达到60%~70%,且对底物cAMP具有更高的亲和力,并确定该酶具有较高生物学活性和稳定性,为该蛋白的晶体学解析及在C.albicans中的生理病理机制研究提供理论依据。For the purpose of preparing high-purity Candida albicans phosphodiesterases 2(PDE2),the genomic DNA of C.albicans was used as a template to amplify the target gene by PCR.The expression plasmid pET28 a-C.albicans pde2 was constructed,and transformed into E.coli BL21 cells to obtain genetically engineered bacteria with high expression stability.The expressed protein from the culture supernatant was consecutively purified by affinity chromatography(Ni-NAT),ion exchange column chromatography(Q-Sepharose)and Sephacryl S200 column chromatography.After that,the hydrolysis characteristics of the purified PDE2(~62 kDa)on single and double substrates were determined by high performance liquid chromatography(HPLC).It was shown that the hydrolysis efficiency of cAMP and cGMP both at 0.4 mmol/L was 60%-70%,and the affinity was higher for cAMP.Furthermore the enzyme had high biological activity and stability.This study provides a theoretical basis for the crystallographic analysis of the protein and for understanding its role in the physiological and pathological mechanism of C.albicans in the future.

关 键 词：白假丝酵母 环核苷酸磷酸二酯酶 表达纯化 高效液相色谱 

分 类 号：TS201.2[轻工技术与工程—食品科学]