Expression and bioconversion of recombinant m- and p-hydroxybenzoate hydroxylases from a novel moderate halophile, Chromohalobacter sp

• Published in: Biotechnology letters 2012-09, Vol.34 (9), p.1687-1692
• Main Authors: Kim, Wonduck, Park, Yu Ri, Im, Seonghun, Kim, Dockyu, Kim, Si Wouk
• Format: Article
• Language: English
• Subjects: Applied Microbiology; Bacteria; Benzoates - metabolism; Biochemistry; Bioconversions. Hemisynthesis; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; biotransformation; Chromohalobacter; Chromohalobacter - enzymology; Chromohalobacter - genetics; Cloning, Molecular; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes; Genetic recombination; Halophiles; heat shock proteins; Heat-Shock Proteins - metabolism; Hydroxybenzoates - metabolism; Kinetics; Life Sciences; Magnesium; Methods. Procedures. Technologies; Microbiology; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - isolation & purification; Mixed Function Oxygenases - metabolism; Original Research Paper; Proteins; Recombinant; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sodium chloride; Solubility; Temperature
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-012-0950-3

p-Hydroxybenzoate hydroxylase (pobA) and m-hydroxybenzoate hydroxylase (mobA) genes, from the moderate halophile Chromohalobacter sp. HS-2, were expressed and characterized. Solubilities of overexpressed recombinant MobA and PobA were enhanced by the induction of the heat-shock proteins DnaJ and DnaK. Each MobA and PobA maintained stable activity under high NaCl concentrations. V max and K m values for MobA with m-hydroxybenzoate were 70 μmol min−1 mg−1 protein and 81 μM, respectively. Similarly, those of PobA with p-hydroxybenzoate as substrate were 5 μmol min−1 mg−1 protein and 129 μM, respectively. The Escherichia coli expression system, including induction of heat shock proteins, was used to convert hydroxybenzoates into protocatechuate (3,4-dihydroxybenzoate) and revealed that resting cells harboring mobA converted 15 mM m-hydroxybenzoate to 15 mM protocatechuate while those harboring pobA converted 50 mM p-hydroxybenzoate to 35 mM protocatechuate at 30 °C, respectively.