A bacterium B2 isolated from the Tianshan glacier of Xinjiang could produce blue pigments.According to 16S rDNA analysis, this isolate belonged to Duganella Genus .Two compounds were separated and purified from the cultivated Duganella B2 , named Blue-Ⅰand Blue-Ⅱ, respectively.From the spectra data of UV, MS and NMR of the compounds, Blue-Ⅰwas confirmed to be deoxyviolacein and Blue-Ⅱ was violacein.Blue-Ⅰand Blue-Ⅱ had the respective molecular weights of 327.2 and 343.2,and showed the characteristic absorption peaks at the respective 560 and 572 nm within the visible light range in ethanol solution.These results will be useful for developing the bioprocess for producing bacterial violacein.
In order to study the effects of ionic surfactants on bacterial luciferase,the cationic surfactant dodecyltrimethylammonium biomide (DTAB) and anionic surfactant sodium dodecylsulfate (SDS) were chosen.For comparison with bacterial luciferase,α-amylase was used since these two enzymes have similar electrostatic potential and charged active sites.After the enzymes were treated with the surfactants,the catalytic properties of bacterial luciferase andα-amylase were assayed,and fluorescence spectroscopy and circular dichroism (CD) were used to analyze the alteration of the protein structure.The results showed that when the DTAB concentration was low,the cationic surfactant DTAB enhanced the enzymatic activities of bacterial luciferase andα-amylase.On the other hand,the anionic surfactant SDS did not alter the enzymatic activity.The main interaction of cationic surfactant DTAB and the negatively charged surface of the proteins was the ionic interaction,which could alter the environment for the enzyme to work when the DTAB/enzyme molar ratio was low.However,at high cationic surfactant concentration,the ionic interaction and hydrophobic interaction might destroy the secondary and tertiary structures of the proteins,leading to the loss of enzymatic activities.
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.
