弗氏柠檬酸杆菌植酸酶的分离纯化及其酶学性质研究  

Purification and properties of Citrobacter freundii phytase

作　　者：罗会颖[1] 石鹏君[1] 李江[1] 王亚茹[1] 姚斌[1] 黄火青[1] 袁铁铮[1] 柏映国[1] 

机构地区：[1]中国农业科学院饲料研究所,北京100081

出　　处：《微生物学报》2006年第1期139-142,共4页Acta Microbiologica Sinica

基　　金：国家"863计划"(2003AA214030);国家"973项目"(2004CB719606)~~

摘　　要：从弗氏柠檬酸杆菌（Citrobacter freundii）中分离纯化了一种植酸酶并进行了酶学性质研究，其反应最适pH为4．0．4．5，最适温度为40℃，在37℃下以植酸钠为底物的Km值为0．85nmol/L，Vmax为0．53IU／（mg·min），具有较好的抗胰蛋白酶的能力。酶蛋白的分子量大小约为45kDa，成熟酶蛋白N端序列为QCAPEGYQLQQVLMM。Phytase （ myo-inositol- 1,2,3,4,5,6- hexakisphosphate phosphohydrolase, EC 3.1.3.26） catalyses the stepwise hydrolysis of phytic acid （myo-inositol hexakisphosphate）. Phytases arc of great commercial importance due to their usage as supplement of food and animal feed, which can cater to nutrition demands and alleviate environmental problems, has been approved by many countries. Although acid phytases have been extensively studied, information regarding the phytases from Citrobacter is limited. In the work presented, a phytase was separated from Citrobacter freundii. After steps of electrophoretic homogeneity by successive ammonium sulfate between 60% and 80% saturation precipitation, DEAE-Sepharose ion-exchange chromatography and gel filtration through Superdex HR 10/30, final gel elution resulted in a 41.3-fold purification and yield of 9.3%. Gel elution is an effective method to purify the protein which contaminated with a few other proteins. The purified preparations were used in subsequent characterization studies. Based on SDSPAGE analysis, the molecular weight of the purified phytase was calculated to be approximately 45.0kDa in monomeric form. The pure enzyme has an optimum pH of 4.0 - 4.5. It was found stable between pH5.0 - 7.0, about 90 % of the enzyme activity was retained at 37℃ for 60min. The phytase has an optimum temperature of 40℃ which was lower than that of other phytases from Aspergillus or E. coli （average 50 - 60℃ ） and was close to the temperature of gastrointestinal tract in animals （37 -40℃ ）. Thus the enzyme is a promising candidate for animal feed applications. Activity of the purified phytase was influenced by changing the reaction temperature. Data showed that the enzyme retained its activity over a long period when stored at 4℃, whereas thermal inactivation studies indicated that the enzyme lost 100% activity after treatment at 60℃ for 4min. The Km values of the phytase for dodecasodium phytate at 37℃ was 0.85nmol/L with a Vmax 0.53IU/（mg·min）. Phyta

关 键 词：弗氏柠檬酸杆菌 植酸酶 酶学性质 

分 类 号：S816.7[农业科学—饲料科学]