Expression, purification and characterization of recombinant 2-(2'-hydroxyphenyl)benzenesulfinase desulfinase from Rhodococcus erythropolis sp. IGTS8
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Catalytic biodesulfurization uses microbes to remove the heterocyclic sulfur from dibenzothiophene-(DBT) without degrading the hydrocarbon fuel value. Rhodococcus erythropolis has been determined to degrade DBT to the final products 2-hydroxybiphenyl and sulfite using four enzymes. The last metabolic step in the pathway is performed by 2-(2’-hydroxyphenyl)benzenesulfinate desulfinase(DszB) and is the rate-limiting step. The gene coding for the DszB from Rhodococcus erthropolis IGTS8 was cloned into the expression vector pTAC-MAT-Tag-2 and then transformed into BL21(DE3) containing pREP4-GroESL. The enzyme was successfully expressed and purified using the ProBondTM Nickel column. Quantification showed a yield of 0.10 mg of DszB from 500 g of wet cells. Fluorimetric studies showed optimal activity at 35 ºC and pH range 8-10. The temperature stability range was 25- 35 º C. Kinetic studies show a Km of 3.75 ± 4.79µM and kcat 0.68 ± 0.15 min-1, respectively. The recombinant HPBS desulfinase showed similar optima temperature and stability as the native enzyme. The kinetic parameters shared similar values with the native enzyme, within experimental error.