大豆下胚轴谷丙转氨酶酶学性质分析  

Enzyme Properties Analysis of Glutamate-Pyruvate Transaminase in Soybean Hypocotyl

作　　者：郭旭舜 林裕添 贺敬东 李昕昊 张尚宏[1] GUO Xu-shun;LIN Yu-tian;HE Jing-dong;LI Xin-hao;ZHANG Shang-hong(School of Life Sciences,Sun Yat-sen University,Guangzhou 510275,China;Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou,510080,China)

机构地区：[1]中山大学生命科学学院,广东广州510275 [2]中山大学中山医学院,广东广州510080

出　　处：《大豆科学》2021年第1期82-88,共7页Soybean Science

基　　金：中山大学本科教学质量工程项目(33000-18842502,33000-18832618)。

摘　　要：大豆是一种重要的固氮农作物,在其种子萌发和初期幼苗生长过程中氨基酸代谢作用较强,表达活性较高的谷丙转氨酶(GPT)。为探明大豆下胚轴谷丙转氨酶的蛋白序列和酶学性质,以指导其在基因工程和农业生产中的应用,本研究通过对豆芽下胚轴蛋白组进行液相色谱串联二级质谱分析,确定大豆下胚轴所表达的GPT序列,利用谷氨酸和丙氨酸与茚三酮反应的显色差异测量GPT活力,并探究其酶学性质。结果显示:以茚三酮比色法测得大豆下胚轴谷丙转氨酶在35℃和pH7.0条件下对于丙酮酸的Km值为0.186 mmol·L-1,对于谷氨酸的Km值为1.05 mmol·L-1;Co2+、Mg2+和Ca2+对大豆谷丙转氨酶有激活效应,EDTA2-、尿素、Mn2+和Fe2+有抑制效应;大豆下胚轴谷丙转氨酶具有活性的温度范围为10~60℃,pH范围为4.0~8.0,最适温度为35℃,最适pH7.0。Soybean(Glycine max) is an important nitrogen-fixing crop. It has strong amino acid metabolism and high expression of vigorous glutamate-pyruvate transaminase(GPT) during seed germination and early seedling growth. In this study, we identified the sequence of the GPT expressed in soybean hypocotyl through liquid chromatography-tandem mass spectrometry(LC-MS/MS) analysis, and described its kinetic characterization to guide its application in genetic engineering and agricultural production. The Km values of this enzyme for glutamate and pyruvate were 1.05 and 0.186 mmol·L-1, respectively, measured by ninhydrin colorimetry based on the chromogenic difference between glutamate and alanine. Co2+, Zn2+, Ca2+ and Mg2+ had promotion effect, while EDTA2-, urea, Mn2+ and Fe2+ had inhibitory effect on the enzyme. The enzyme was active in the temperature range between 10 and 60 ℃, with the optimum temperature of 35 ℃, and active in the pH range between 4.0 and 8.0, with the optimum pH7.0, measured by separating glutamate and alanine through paper chromatography and calculating the enzyme activity.

关 键 词：大豆 下胚轴 谷丙转氨酶 酶学性质 液相色谱串联二级质谱 茚三酮比色法 

分 类 号：S565.1[农业科学—作物学]