木豆种子酸性磷酸酯酶AcPase I的部分纯化和动力学特性  

Partial purification and dynamics characteristics of acid phosphatase I from pigeon pea seed

作　　者：杨杰[1] 董登峰[1] 王永峰[2] 

机构地区：[1]广西大学农学院,南宁530005 [2]广西农业科学院广西作物遗传改良生物技术重点开放实验室,南宁530007

出　　处：《广西农业科学》2007年第4期386-390,共5页Guangxi Agricultural Sciences

基　　金：广西大学博士启动基金项目(DD160008)

摘　　要：以萌发的木豆种子为材料,经过匀浆、硫酸铵分部（20%-60%饱和度）沉淀、DEAE-Sephadex A25离子交换与凝胶柱层析,可得到两个酶活力峰AcPase I和AcPase。将AcPase I过HA和ConA-Sepharose 4B柱层析后,被纯化了51.3倍,比活性为10.80U/mg。AcPase I经PAGE和SDS-PAGE分析都呈现单一蛋白酶带,证明Ac-Pase I是单亚基蛋白酶,其亚基分子量约为32kDa。以对硝基苯磷酸作底物时,AcPase I最适pH为4.0,最适温度为50℃,Km为2.48mmol/L,专一性常数为1.47U/mM.mg,AcPase I对焦磷酸有最小的米氏常数0.15mmol/L,有最大的专一性常数74.63 U/mM.mg。因此,AcPase I的这些动力学特性可为今后进一步研究提供理论依据。The germinating seeds of pigeon pea were used as material, and two isoenzymes （AePase Ⅰand AcPase Ⅱ） of acid phosphatase were separated and purified from grinding of pigeon pea seeds, ammonium sulfate frae tion precipitation （20%-60% saturation）, DEAE-Sephadex A25 column chromatography. AcPase Ⅰ was purified up to 51.3 times with a final specific activity of 10.80U/mg through Hydroxyapatite column chromatography and ConA Sepharose 4B column chromatography. AcPase Ⅰwas of a single electrophoretic band in PAGE and SDSPAGE, respectively, it showed that AcPase Ⅰwas a single subunit protcase, and the molecular mass was 32kDa. The optimum pH of AePase Ⅰ was 4.0 when pNPP was used as substrate, and the optimum temperature was 50 ℃, the specificity constants （Vmax/Km） was 1.47U/mM·mg and Miehaelis-Menten Kinetics （Kin） was 2.48mmol/ L. Moreover, the maximal specificity constant （Vmax/Km） of AcPase I to pyrophosphate （PPi） was 74. 63 U/mM·mg and the minimal Km was 0.15 mmol/L. Therefore, these dynamics characteristics of AePase 1 would provide the theory basis for further research in the future.

关 键 词：木豆 酸性磷酸酯酶 纯化 动力学 特性 

分 类 号：S330.2[农业科学—作物遗传育种]