Expression, Purification and Characterization of Recombinant 2-(2'-hydroxyphenyl)benzenesulfinase desulfinase from Rhodococcus erythropolis sp. IGTS8




Harper, Leanne T.

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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.



Desulfurization, Rhodococcus erythropolis IGTS8, Recombinant expression system, GroESL, Chaperonine proteins


Harper, L. T. (2011). <i>Expression, purification and characterization of recombinant 2-(2'-hydroxyphenyl)benzenesulfinase desulfinase from Rhodococcus erythropolis sp. IGTS8</i> (Unpublished thesis). Texas State University-San Marcos, San Marcos, Texas.


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