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,C23...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.展开更多
文摘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.
