Expression and bioconversion of recombinant m- and p-hydroxybenzoate hydroxylases from a novel moderate halophile, Chromohalobacter sp.
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.
Department of Environmental Engineering, BK21 Team for Biohydrogen Production, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, South Korea.
Wonduck
W
Kim
Yu Ri
YR
Park
Seonghun
S
Im
Dockyu
D
Kim
Si Wouk
SW
Kim
D001565:Benzoates / Q000378:metabolism*; D055353:Chromohalobacter / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000378:metabolism; D062385:Hydroxybenzoates / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D001565:Benzoates; D006360:Heat-Shock Proteins; D062385:Hydroxybenzoates; D011994:Recombinant Proteins; C009091:protocatechuic acid; D006899:Mixed Function Oxygenases