THE GENE CLONING, OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION OF DIBENZOTHIOPHENE MONOOXYGENASE AND DESULFINASE FROM GORDONIA ALKANIVORANS RIPI۹۰A

The biodesulfurization (BDS) of sulfur compounds in fossil fuels is a process to reduce sulfur dioxide emissions that cause environmental pollution. Gordonia alkanivorans RIPI۹۰A is able to convert dibenzothiophene, an organic sulfur compound in petroleum, to ۲-hydroxybiphenyl (۲-HBP) in ۴S pathway. In this study, (DBT), DszA and DszB, DBT sulfone monooxygenase, and desulfinase were respectively isolated from G. alkanivorans RIPI۹۰A. PCR amplified fragments were obtained by using primers designed based on known sequences from G. alkanivorans RIPI۹۰A. They are identified as dszA and dszB and have shown high similarity compared to Rhodococcus erythropolis IGTS۸ (۸۸% for dszA and ۸۸% for dszB). Subsequently, dszA and dszB genes were expressed under the control of T۷ promoter in Escherichia coli. The recombinant proteins were purified to achieve homogeneity using Ni-agarose column chromatography. The molecular mass of the purified DszA and DszB were determined to be ۵۱.۹ and ۳۹.۲ kDa respectively by using SDS-polyacrylamide gel electrophoresis. DszA showed a Km of ۰.۱۴±۰.۰۰۵ mM and a maximal velocity of ۰.۰۰۴±۰.۰۰۰۴ mM/min. DszB showed a wide substrate range in a way that all aromatic sulfonates compounds acted as its substrate; as it seemed the active site was suitable for the sulfonated aromatic rings. The Km and Vmax values of DszB were calculated to be ۱.۸۱±۰.۰۲ mM and ۶.۵۵ ± ۰.۰۰۵ µM/min respectively using ۴-Amino-۳-hydroxy-naphthalene-sulfonic acid as a substrate

کلیدواژه ها:

DszA ، DszB ، Purification ، Gordonia alkanivorans RIPI۹۰A ، Biodesulfurization