罗伊氏乳杆菌中甘油脱水酶的催化特性及原位再激活  

Characteristics and in situ Reactivation of Glycerol Dehydratase in Lactobacillus reuteri

作　　者：马会亮[1] 陈国[1] 赵珺[1] 

机构地区：[1]华侨大学化工学院生物技术与工程系,厦门361021

出　　处：《应用与环境生物学报》2013年第1期30-36,共7页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(20906035);华侨大学中央高校基本科研业务费项目(JB-JX1002)资助~~

摘　　要：对罗伊氏乳杆菌(Lactobacillus reuteri)全细胞和粗酶液催化转化甘油和1,2-丙二醇进行比较,分析其底物转化特性;使用有机溶剂制备通透细胞,外源添加辅酶B12和ATP,探索L.reuteri胞内甘油脱水酶再激活机制.L.reuteri胞内存在依赖于辅酶B12的甘油脱水酶,1,2-丙二醇不会使甘油脱水酶全酶失活,而甘油会使全酶失活;其最适温度和最适pH分别为37℃和6.2;获得了制备L.reuteri通透细胞的最佳条件;向通透细胞添加辅酶B12和ATP均能促进甘油脱水酶催化甘油转化,辅酶B12仅与甘油脱水酶单酶结合形成具催化活性的全酶,而ATP能协助失活辅酶脱离全酶和失活辅酶再激活过程.L.reuteri与Klebsiella pneumoniae类似,胞内存在甘油脱水酶再激活机制,但来源于二者的甘油脱水酶氨基酸序列同源性较低.来源于L.reuteri的甘油脱水酶是典型的依赖于辅酶B12的甘油脱水酶,尽管其序列同源性与来源于K.pneumoniae、Citrobacter freundii的甘油脱水酶差异较大,但在胞内同样存在甘油脱水酶再激活机制,可利用该机制实现胞内甘油脱水酶原位再激活,进而达到重复利用的目的.For understanding the catalysis properties and in situ reactivation of glycerol dehydratase in Lactobacillus reuteri, the conversions of glycerol and 1,2-propanediol by whole cell of L. reuteri and crude enzyme supernatant were compared to analyze substrate inhibition characteristics of dehydratase. Some organic solvents were used to improve the cell permeability, which did little harm to cells and dehydratase. Coenzyme Bt2 and ATP were added to treated cell to explore the in vivo reactivation mechanism of dehydratase in L. reuteri. The glycerol dehydratase in L. reuteri was coenzyme B12 dependent, which would be inactivated by glycerol rather than 1,2-propanediol. Its optimal temperature and pH were 37 ~C and 6.2, respectively. The conditions for improving the cell permeability were obtained. The glycerol conversion was increased by adding coenzyme B12 and ATP. The coenzyme B^2 was combined with apoenzyme to form holoenzyme which had catalysis activity, but the ATP can assist the detachment of inactive coenzyme B^z from the holoenzyme and transformation of inactive coenzyme B^2 to an active one. The reactivation mechanism of glycerol dehydratase existed in L. reuteri like K. pneumonia, but their sequence homology was low. The glycerol dehydratase from L. reuteri is a typical coenzyme B12 dependent enzyme. There is reactivation mechanism of glycerol dehydratase in vivo, though it shares low similarities in sequences with C. freundii and K. pneumonia. This mechanism can be used to achieve the reusing of glycerol dehydratase in vivo. Fig 9, Ref 23

关 键 词：罗伊氏乳杆菌 甘油脱水酶 辅酶B12 3-羟基丙醛 

分 类 号：Q78[生物学—分子生物学]