ABSTRACT
Aims@#Metal transcriptional regulators controlled the regulation of metal ion homeostasis in bacteria genera. Cd(II)/Pb(II) transcriptional regulator is one of the member of MerR family found in Alcaligenes faecalis SF-S1-60 (PbrT-AF). @*Methodology and results@#The PbrT-AF gene with 432 bp open reading frame was successfully isolated from genomic DNA of A. faecalis using polymerase chain reaction (PCR) analysis. This gene was phylogenetically grouped with A. alcaligenes species using PHYLIP version 3.69 by the neighbor-joining method with 1000 bootstrap replicates. Phylogeny analysis shows that these proteins have distinct amino acids compared to Cd(II)/Pb(II) regulators from different species. The structure of PbrT-AF shows similar conformation with other members of MerR family using MODELLERv9.17. We also demonstrated that the expression of Pbrt-AF in Escherichia coli BL21 were able to increase the bacteria tolerance towards Pb up to 1000 ppm. @*Conclusion, significance and impact of study@#This result suggests that PbrT-AF promotes cell adaptation and tolerance towards Pb toxicity.
ABSTRACT
Background: Benzaldehyde dehydrogenase [BZDH] is encoded by the xylC that catalyzes the conversion of benzaldehyde into benzoate in many pathways such as toluene degradation
Objectives: In this study, the xylC gene from Rhodococcus ruber UKMP-5M was expressed in Escherichia coli, purified, and characterized
Materials and Methods: The xylC was amplified and cloned in E. coli. The recombinant plasmid pGEMT-xylC was digested by NdeI and HindIII to construct plasmid pET28b-xylC and transformed in E. coli BL21 [DE3]. Expression of the recombinant protein was induced by 1 mM isopropyl beta-D-thiogalactoside [IPTG] at 37[degree]C. The BZDH was purified by ion exchange chromatography, in which the product was an NAD-dependent enzyme using benzaldehyde as a substrate for enzyme characterization. The end metabolite was identified via gas chromatography mass spectrometry [GC-MS]
Results: The recombinant BZDH is 27 kDa, purified by ion exchange chromatography. The activity of BZDH was 9.4 U.microL[-1] The optimum pH and temperature were 8.5 and 25[degree]C, respectively. The Michaelis constant [K[m]] and maximum velocity [V[max]] were 4.2 mM and 19.7 U.mL[-1], respectively. The metabolite of BZDH was benzene carboxylic acid as determined by GC-MS analysis
Conclusions: BZDH has the ability to degrade benzaldehyde to less toxic compounds. The BZDH is a critical enzyme for the degradation of aromatic hydrocarbons in Rhodococcus sp. The BZDH from R. ruber UKMP-5M is showed similar function with other aldehyde dehydrogenases