Specificity Studies of Recombinant 2-(2'-Hydroxyphenyl) Benzenesulfinate Desulfinase (DszB) from Nocardia Asteroides A3H1 and Rhodococcus Erythropolis IGTS8
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Biodesulfurization is a process that uses bacteria that contain enzymes capable of selectively removing sulfur from petroleum. The dsz pathway is well studied and capable of removing sulfur from dibenzothiophene (DBT). A desulfinating enzyme, 2-(hydroxyphenyl)benzenesulfinate desulfinase (DszB) catalyzes the final step of the dsz pathway, cleaving the sulfinate bond and releasing the sulfur without destroying the carbon backbone of the compound. The dsz genes from Rhodococcus erythropolis IGTS8 and Nocardia asteroides A3H1 were previously cloned into pTAC-MAT-Tag-2 and transformed into BL21(DE3) containing pREP4-GroESL. The plasmid pREP4-GroESL contains genes than encode the GroES and GroEL, chaperones that participate in the proper folding of DszB. Structural and bioinformatic studies of DszB were conducted on the sequence of amino acids from 180 to 210. Amino acids A195 and A200 were selected to be mutated to arginine and were hypothesized to be important in determining the enzyme specificity for Nocardia asteroides A3H1. Site-directed mutagenesis of the dsz gene was conducted to create DszB-A3H1-A200R. Recombinant enzymes were purified with nickel affinity chromatography, placed in a fixed timed assay with appropriate substrate, and coupled with sulfite oxidase to study enzyme specificity. The desulfinase enzyme DszB-A3H1-A200R was characterized with fluorescent studies that included kinetic, pH, temperature, and temperature stability. Fluorimetric studies show that the mutation increased the enzyme temperature stability and increased the ability to desulfinate at higher pH values. The kinetic data showed a km of 3.71 ± 1.54 μM and kcat of 1.1 ± 0.10 min-1.The specificity of DszB-A3H1-A200R showed a change in specificity, preferring 2-(2’-hydroxyphenyl)enthen-1-sulfinate (HBES) over benzenesulfinate (BS).
CitationCromwell, D. N. (2013). Specificity studies of recombinant 2-(2'-Hydroxyphenyl) benzenesulfinate desulfinase (DszB) from nocardia asteroides A3H1 and rhodococcus erythropolis IGTS8 (Unpublished thesis). Texas State University, San Marcos, Texas.