建立转酮酶酶活测定方法及应用  

Establish and application of the assay for determining transketolase activity

作　　者：李胜营[1,2] 朱欣娜[2] 毛小琴[1,3,4] 

机构地区：[1]昆明理工大学医学院,云南昆明650500 [2]中国科学院天津工业生物技术研究所,天津300308 [3]云南省第一人民医院,昆明理工大学附属医院,云南昆明650500 [4]云南省临床微生物学与分子研究中心,云南昆明650500

出　　处：《中国微生态学杂志》2016年第7期840-845,共6页Chinese Journal of Microecology

基　　金：国家自然科学基金资助项目(31300089);云南科技计划面上项目(2014NS247)

摘　　要：目的转酮酶是非氧化磷酸化戊糖途径中的关键酶。5-磷酸木酮糖是分析测定转酮酶酶活性的底物,然而因该底物难以化学合成,生产成本高,从而导致厂家停止生产,目前市场上无法得到。因此有必要建立起新的转酮酶酶活测定方法。方法(1)将酿酒酵母的木酮糖激酶基因(XKS1)克隆于pET30a载体。(2)纯化木酮糖激酶。(3)建立酶偶联反应,以木酮糖为底物,将其转化为5-磷酸木酮糖,用于转酮酶酶活的测定。结果 (1)成功地将XKS1基因克隆于pET 30a得到质粒pXZ-X004。(2)质粒pXZ-X004诱导表达产生C端His-tag标签的XK,用Ni柱分离得到纯化的木酮糖激酶(XK)。(3)纯化的XK活性为21.0U/mg protein,当用12.5%的甘油保存于-80℃环境1个月,仍有74%的活性。(4)用纯化的XK建立了转酮酶(TK)酶活测定新方法,当TK酶活低至0.008 75U时仍能够利用新方法检测。结论本实验通过克隆和表达XKS1基因,成功地建立了TK酶活性测定的新方法,并应用于木糖代谢工程菌株TK酶活的测定,为医学和生态学领域中TK酶活的分析奠定了基础。Objective To establish a new method for determining transketolase activity. Methods （1） Xylulose kinase gene （XKS1） from Saccharomyces cerevisiae was cloned into vector pET30. （2） Xylulose kinase （XK） was expressed and purified. （3） An enzyme coupling reaction was performed to convert xylulose into xylulose-5-phostate which was used for determining the activity of transketolase. Results （1） The XKS1 gene was successfully cloned into pET30a, resulting in pXZ-X004. （2） XK from pXZ-X004 was expressed, which was an enzyme with C-terminal His tag. The purified XK was obtained by Ni separating column. （3） The activity of XK was 21.0 U/mg protein. When the purified XK protein was preserved in 12.5% of glycerol at -80 ℃, it remained 74% of activity after a month. （4） A new method for determining transketolase activity was created. When the TK enzyme activity even is 0.00875 U, it can be detected by new methods. Conclusion A new method for the determination of TK activity was successfully established, and applied for detecting the TK activity in the xylose metabolic engineering strains.

关 键 词：转酮酶 酶活测定 木酮糖激酶 

分 类 号：Q939.97[生物学—微生物学]