Cloning and characterisation of four catA genes located on the chromosome and large plasmid of Pseudomonas putida ND6

Background: Although the functional redundancy of catechol 1,2-dioxygenase (C12O) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C12O genes. Results: In this study, four novel C12O genes, designated catA, catAI, catAII and catAIII, in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C12O isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatAI and CatAIII were maximally active at a temperature of 45°C, whereas a higher optimum temperature was observed for CatAII at a temperature of 50°C. CatAI exhibited superior temperature stability compared with the other three C12O isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatAI and CatAII, whereas that of CatAIII was lower. Significantly, among metal ions tested, only Cu2+ substantially inhibited the activity of these C12O isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity clearly indicated that the redundancy of the catA genes has increased the levels of C12O. Conclusion: The results clearly imply that the redundancy of catA genes increases the available amount of C12O in P. putida ND6, which would be beneficial for survival in challenging environments.

Enhancement of biodegradation potential of catechol 1, 2-dioxygenase through its immobilization in calcium alginate gel

Background In biodegradation processes free enzymes often undergo deactivation. Thus, it is very important to obtain highly stable enzymes by different methods. Immobilization allows for successful stabilization of many multimeric enzymes by increasing the rigidity of the enzyme structure. This study aimed to evaluate some environmental factors that affect catechol 1,2-dioxygenase from Stenotrophomonas maltophilia KB2 immobilized in alginate hydrogel. The goal of the present work was to improve the functional stability of the enzyme by increasing its structural rigidity. Results Immobilization yield and expressed activity were 100% and 56%, respectively. Under the same storage conditions, the activity of the immobilized enzyme was still observed on the 28th d of incubation at 4°C, whereas the free enzyme lost its activity after 14 d. The immobilized enzyme required approximately 10°C lower temperature for its optimal activity than the free enzyme. Immobilization shifted the optimal pH from 8 for the soluble enzyme to 7 for the immobilized enzyme. The immobilized catechol 1,2-dioxygenase showed activity against 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol, and 3,5-dichlorocatechol. The immobilization of the enzyme promoted its stabilization against any distorting agents: aliphatic alcohols, phenols, and chelators. Conclusions The entrapment of the catechol 1,2-dioxygenase from S. maltophilia KB2 has been shown to be an effective method for improving the functional properties of the enzyme. Increased resistance to inactivation by higher substrate concentration and other factors affecting enzyme activity as well as broadened substrate specificity compared to the soluble enzyme, makes the immobilized catechol 1,2-dioxygenase suitable for the bioremediation and detoxification of xenobiotic-contaminated environments.

Metabolism of 3-chlorobenzoate by a Pseudomonas (diff) spp.

Pseudomonas (diff) spp. was isolated from a complex petrochemical sludge, using benzoate as the sole source of carbon. The organism could metabolize 3-chlorobenzoate, releasing approximately 30% of organically bound chloride. 3-Chlorodihydrodihydroxybenzoate and 3-chlorocatechol were confirmed as pathway intermediates by mass spectral and HPLC analysis. About 3-fold higher levels of catechol 1,2-oxygenase were detected in cells grown on 3-chlorobenzoate as compared to that of benzoate. 3-Chlorocatechol inhibited the catechol 1,2-oxygenase activity, when used as assay substrate. A 15-fold purified catechol 1,2-oxygenase had a Km of 0.37 mumole and Vmax of 2.3 with 3-chlorocatechol. Catechol gave Km of 0.2 mumole and Vmax of 40, suggesting that 3-chlorocatechol is not metabolised further and hence blocks the metabolic pathway for 3-chlorobenzoate degradation. In contrast catechol 1,2-oxygenase was not inhibited by 4-chlorocatechol and probably is an intermediate for the total/complete degradation of 3-chlorobenzoate (approx. 30%).