Penicillin acylase catalyzed synthesis of penicillin-G from substrates anchored in cyclodextrins.

Penicillin acylase (EC 3.5.1.11) catalyses the condensation of phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA) to form benzylpenicillin (BP). Both PAA and 6-APA were found to form host-guest complexes with beta-methylcyclodextrin (beta m-CD) and gamma-cyclodextrin (gamma-CD) respectively. The rate of the reaction catalyzed by the enzyme remained unaffected if one of the substrates used was in the cyclodextrin complexed form. However, in this case, the reaction lasted longer and yielded about 20 per cent more products compared to the condensation reaction involving only uncomplexed substrates. There was distinct increase in the rate of formation of the antibiotic, if both substrates used are in CD-complexed form.

Affinity properties of phosvitin: interaction of phosvitin with serine hydroxymethyl transferase.

The affinity of phosvitin with serine hydroxymethyl transferase (SHMT), an acidic multi-subunit protein, was evaluated by measurements of enzyme activity, sedimentation velocity, steady-state fluorescence, circular dichroism and kinetic thermal stability. While the presence of phosvitin had no effect on the SHMT activity, the sedimentation coefficient of SHMT increased from 8.7 S to 12.5 S suggesting the formation of a complex at a SHMT:phosvitin molar ratio of 2:1. Based on steady-state fluorescence quenching measurements an association constant of 2.4 +/- 0.2 x 10(5) M-1 at 25 degrees C was obtained for the interaction of phosvitin with SHMT. The temperature dependency of the association constant in the range 15-35 degrees C suggests the involvement of ionic forces in the interaction. The thermal inactivation of SHMT followed first order kinetics. In the presence of phosvitin the rate constant decreased and half time increased. The circular dichroism measurements suggest that phosvitin interaction does not involve pyridoxal phosphate binding domain of the enzyme. Although minor changes in the secondary structure of the enzyme were observed, the environment around aromatic amino acids did not change significantly.

Evidence for beta-galactosidase catalyzed hydrolysis of paranitrophenyl-beta-D-galactopyranoside anchored in cyclodextrins.

The absorption maximum of p-nitrophenol upon mixing with molar equivalents of alpha-cyclodextrin (alpha-CD) or hydroxypropyl-beta-cyclodextrin (HPB) showed nearly 5 nm shift to the longer wavelength region, indicative of complex formation, while beta-cyclodextrin (beta-CD), gamma-cyclodextrin (gamma-CD) and hydroxyethyl-beta-cyclodextrin (HEB) produced only a marginal shift of about 1-2 nm, suggestive of a weaker interaction. It has been shown by circular dichroism spectral studies that the aglycon part of p-nitrophenyl-beta-D-glycoside (PNPG) is also encapsulated by alpha-cyclodextrin. The encapsulated form of PNPG could be hydrolyzed by beta-galactosidase, the temperature and pH-optima for hydrolysis of anchored substrates being essentially identical to that of free substrate. However, small but consistent increase in Km values were obtained for alpha-cyclodextrin-, HEB- and HPB-anchored substrates. The kcat values also registered an increase for the HEB- and HPB-anchored substrates. However, there was no increase in kinetic efficiency (kcat/Km) of enzyme. The inhibition noted at higher concentrations of HEB- and gamma-cyclodextrin-anchored PNPG but not with o-nitrophenyl-beta-D-galactoside (ONPG)-cyclodextrin mixture suggests that PNPG-cyclodextrin complexes were responsible for the inhibition. Taken together, these results suggest that the enzyme catalyses the hydrolysis of anchored substrates.

Influence of detergents and pH on the isolation, purification and molecular properties of alpha-galactosidase-C2 from germinating guar (Cyamopsis tetragonolobus).

alpha-Galactosidase was isolated from germinating guar. The extract also contained small amounts of alpha-mannosidase and beta-mannosidase activities. The fractionation of the enzyme extract with ammonium sulphate (75% saturation) resulted in the appearance of all the three enzymes in a floating lipid complex. The inclusion of detergents such as Triton X-100 and sodium deoxycholate in the extraction medium failed to prevent the appearance of these enzymes in the floating lipid complex. However, by using acetone powder of the seedlings, alpha-galactosidase could be sedimented with ammonium sulphate. The presence of detergents in the extraction medium affected the molecular properties of the enzyme. Using a set of carefully selected conditions alpha-galactosidase was purified to apparent homogeneity. Analytical ultracentrifugation and gel filtration studies of the purified enzyme showed association-dissociation phenomenon as a function of pH and temperature. The effect of pH on the association-dissociation indicates the predominance of electrostatic interactions in the association of subunits.