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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.

The purpose of this study was to investigate the influence of pH on properties and stability of oil-in-water emulsions using pectin as an emulsifier and zein as an auxillary emulsifier. Primary emulsions were prepared by mixing rice bran oil with anionic polysaccharide, pectin, at pH 4 or 7. Zein solution was then added in order to prepare secondary emulsions. Final concentration of secondary emulsions was 20% (w/w) rice bran oil, 2% (w/w) pectin and 0.5% (w/w) zein. The results demonstrated that emulsions prepared at pH 4 were smaller in size, higher viscosity, and more stable after stability test than those prepared at pH 7. Light microscopic images showed that, at pH 7, emulsion droplets were aggregated. At pH 7, both pectin and zein represented as negative charge and no complex was formed, leading to the less stable emulsions. Below the isoelectric point (pH 5.5) of zein, i.e. at pH 4, zein molecules represented as positive charge and pectin represented as negative charge, resulting in a light cross-linking polymer network. It is possible that a low concentration of zein helps to stabilize the emulsions by improving the rigidity of coated layer on oil droplets, thus reducing the depletion flocculation to occur. The stabilizing effect in presence of zein, at pH 4, is attributed to a pectinzein complex formation at the interface.

As a new processing method for the production of modified starch, microwave heating was applied to native arrowroot starch. The effect of processing parameter on the sustained release properties of the obtained modified starches was investigated. The modified starch was blended with a model drug, theophylline, and then compressed into tablet using hydraulic press at a pressure of 19.6 kN for 30 s. The physical properties as well as drug release behavior of the compressed tablets were investigated. The results showed that the physical properties of different modified starches were about the same. The modified arrowroot starch displayed better sustained release properties than native starch. The modified arrowroot starch, which subjected to microwave heating using high level of water content, long heating time and high microwave power, demonstrated promising properties as hydrophilic matrix excipients for sustained release tablets.