ABSTRACT
3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from Pseudomonas aeruginosa (PaDHPAO) was overexpressed in Escherichia coli and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM-1cm-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a Km for DHPA of 58 ± 8 µM and a kcat of 64 s-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(k'cat) versus the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (Ea) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology.
Subject(s)
Dioxygenases/metabolism , Pseudomonas aeruginosa/enzymology , Aldehydes/chemistry , Aldehydes/metabolism , Ascorbic Acid/chemistry , Ascorbic Acid/metabolism , Catalase/chemistry , Catalase/metabolism , Escherichia coli/enzymology , Hydrogen-Ion Concentration , Kinetics , Magnetic Resonance Spectroscopy , Models, Molecular , Substrate Specificity , Superoxide Dismutase/chemistry , Superoxide Dismutase/metabolism , TemperatureABSTRACT
The aim of this study was to investigate the effect of high-pressure homogenization on the droplet size and physical stability of different formulations of pectin-zein stabilized rice bran oil emulsions. The obtained emulsions, both before and after passing through high-pressure homogenizer, were subjected to stability test under environmental stress conditions, that is, temperature cycling at 4 °C/40 °C for 6 cycles and centrifugal test at 3000 rpm for 10 min. Applying high-pressure homogenization after mechanical homogenization caused only a small additional decrease in emulsion droplet size. The droplet size of emulsions was influenced by the type of pectin used; emulsions using high methoxy pectin (HMP) were smaller than that using low methoxy pectin (LMP). This is due to a greater emulsifying property of HMP than LMP. The emulsions stabilized by HMP-zein showed good physical stability with lower percent creaming index than those using LMP, both before and after passing through high-pressure homogenizer. The stability of emulsions after passing through high-pressure homogenizer was slightly higher when using higher zein concentration, resulting from stronger pectin-zein complexes that could rearrange and adsorb onto the emulsion droplets.
