Abstract:

AtSHM1 gene which encoding SHMT protein was cloned  in mitochondria from the leaf of Arabidopsis thaliana, inserted it into pET\|26b (+) expression vector, and successfully expressed in Escherichia coli. After optimizing the expressive condition, the soluble AtSHMT protein was obtained. This protein was successfully purified by affinity chromatography method.  Km and Vmax value of this enzyme were 121 μmol and 21.8 μmol/L·min, respectively. AtSHMT could catalyze DL\|3\|Phenylserine to produce benzaldehyde, which had strong absorbance at 279 nm. We measured the light absorption value of the reaction product, and tested the enzyme activity of AtSHMT according to standard curve of benzaldehyde. Therefore this research developed a simple, fast and safe method to measure the enzyme activity of AtSHMT.

Key words: serine hydroxymethyltransferase, affinity chromatography, enzyme assay

摘要：

从拟南芥叶片中克隆了定位于线粒体的拟南芥丝氨酸羟甲基转移酶基因(AtSHM1)，将其插入到pET\|26b(+)表达载体，并在大肠杆菌中成功表达。优化表达条件之后，获得可溶性AtSHMT蛋白。用亲和色谱法成功分离纯化该蛋白，计算得到该酶酶学常数Km和Kmax分别为121 μmol和21.8 μmol/L·min。利用该酶催化DL\|3\|苯基丝氨酸裂解的产物苯甲醛在279 nm波长处有强烈吸收的性质，测定反应产物的吸光值，根据苯甲醛标准曲线测得AtSHMT酶活，由此建立了一种测定真核生物丝氨酸羟甲基转移酶酶活的一种简便、快速、安全的方法。

关键词: 丝氨酸羟甲基转移酶；亲和色谱；酶活测定