Hunter, Farley Allen. Purification and characterization of the hydroxylaminobenzoate lyase from pseudomonas pickettii YH105, cloned in escherichia coli. Retrieved from https://doi.org/doi:10.7282/T3G44QNJ
DescriptionThe hydroxylaminolyase enzyme of Pseudomonas pickettii YH105 (now Ralstonia pickettii) removed the hydroxylamino group from p-hydroxylaminobenzoate with the concomitant formation of protocatechuate. In prior work, Brian A. Koller transferred the YH105 gene coding for hydroxyaminolyase into Escherichia coli now identified as BAK100. Although transferred, the hydroxyaminolyase enzyme was not characterized with respect to protein size and activity.
Utilizing samples of BAK100, the bacteria was grown on Luria-Bertani broth, induced with isopropyl β-D-1-thiogalactopyranoside (IPTG), separated by centrifugation, washed and weighed. After suspension in morpholinopropane sulfonic acid (MOPS) buffer, the enzyme was released from the crude cells by processing through a French press. Following centrifugation to remove non-target cell fragments, protamine sulfate treatment was used to remove RNA. The resulting solution was concentrated and non-target protein removed utilizing ammonium sulfate treatment prior to gradient DEAE and Sepharose 6L-6B gel filtration. Other purification techniques evaluated and discarded included dialysis, hydroxylapatite treatment, polyethylene glycol precipitation, and pH precipitation. Overall, the protein was purified relative to the crude cell extract, increasing by a factor of 2.7. The purification steps resulted in a substantial loss of activity. The overall yield was only 5%.
Several SDS electrophoresis gels all showed multiple prominent bands preventing the determination of enzyme size. Kinetic studies utilizing HPLC to quantifying protocatechuate formation provided a Km of 0.079 mM and Vmax of 0.16 µmol/min·mg for protamine sulfate treated crude cell extract.
Substrates other than p-hydroxylaminobenzoate were tested to evaluate enzyme specificity including m-hydroxylaminobenzoate, 3-methyl-4-hydroxylaminobenzoate and
p-hydroxylamino phenyl acetic acid. Only 3-methyl-4-hydroxylaminobenzoate showed slight conversion, suggesting a high level of enzyme specificity.