邻苯二酚2,3-双加氧酶在恶臭假单胞杆菌整细胞催化中的酶活检测方法  

A simple assay of catechol 2,3-dioxygenase activity in whole-cell catalysis of Pseudomonas putida mt-2

作　　者：张建峰[1] 苏凤宜[1] 邢新会[1] 

机构地区：[1]清华大学化学工程系生物化工研究所

出　　处：《化工学报》2008年第2期450-455,共6页CIESC Journal

基　　金：国家自然科学基金项目(20676071,20336010)~~

摘　　要：Catechol 2,3-dioxygenase(C23O)is the key enzyme of aromatic substance degradation by Pseudomonas sp..In order to establish a simple assay of C23O activity during the whole-cell catalysis of Pseudomonas putida mt-2,C23O was induced by utilizing sodium benzoate acid as the sole carbon source,and its activity was determined in whole cells by the amended protocol of pure enzyme assay.After suspending the cells with potassium phosphate buffer,the substrate was added and the accumulation of 2-hydroxymuconic semialdehyde was measured by a UV757CRT spectrophotometer at 375 nm.The activity of C23O was evaluated by the climbing slope of time course curve of the UV absorption.By this means,the Km for catechol and C23O in whole cells was 34.67 μmol·L-1,while Vmax was 0.29 μmol·min-1·(mg dry cell)-1,both of which differed from those for pure enzyme by 2—3 orders of magnitude.To eliminate the cell wall barrier for substrate permeation,a cationic surfactant,n-dodecyltrimethylammonium bromide,was used to pre-treat the cells.With 0.1 g·L-1 dodecyl trimethyl ammonium bromide(DTAB) treated for 30 min,the maximum C23O activity could be achieved,which was consistent with the result of treated cells by beads milling.In the present study,a feasible and simple method was put forward for the apparent enzyme activity assay intracells which could be conveniently applied to the whole-cell biocatalysis or to environmental bioremediation.Catechol 2, 3-dioxygenase （C23O） is the key enzyme of aromatic substance degradation by Pseudomonas sp.. In order to establish a simple assay of C23O activity during the whole-cell catalysis of Pseudomonas putida mt-2, C23O was induced by utilizing sodium benzoate acid as the sole carbon source, and its activity was determined in whole cells by the amended protocol of pure enzyme assay. After suspending the cells with potassium phosphate buffer, the substrate was added and the accumulation of 2-hydroxymuconic semialdehyde was measured by a UV757CRT spectrophotometer at 375 nm. The activity of C23O was evaluated by the climbing slope of time course curve of the UV absorption. By this means, the Km for catechol and C23O in whole cells was 34.67 μmol · L^-1 , while Vmax was 0. 29 μmol· min^-1 · （mg dry cell）^-1, both of which differed from those for pure enzyme by 2-3 orders of magnitude. To eliminate the cell wall barrier for substrate permeation, a cationic surfactant, n-dodecyltrimethylammonium bromide, was used to pre-treat the cells. With 0.1 g · L^-1 dodecyl trimethyl ammonium bromide （DTAB） treated for 30 min, the maximum C23O activity could be achieved, which was consistent with the result of treated cells by beads milling. In the present study, a feasible and simple method was put forward for the apparent enzyme activity assay intracells which could be conveniently applied to the whole-cell biocatalysis or to environmental bioremediation.

关 键 词：邻苯二酚2  3-双加氧酶 整细胞催化 十二烷基三甲基溴化铵 环境修复 

分 类 号：TQ033[化学工程] X172[环境科学与工程—环境科学]