Curcumin (diferuloyl methane) is the physiologically and pharmacologically active component of turmeric (Curcuma longa L.). Solubility and stability of curcumin are the limiting factors for realizing its therapeutic potential. β-Lactoglobulin (βLG), the major whey protein, can solubilize and bind many small hydrophobic molecules. The stability of curcumin bound to βLG in solution is enhanced 6.7 times, in comparison to curcumin alone (in aqueous solution). The complex formation of curcumin with βLG has been investigated employing spectroscopic techniques. βLG interacts with curcumin at pH 7.0 with an association constant of 1.04 ± 0.1 × 10(5) M(-1) to form a 1:1 complex at 25 °C. Entropy and free energy changes for the interaction derived from the van't Hoff plot are 18.7 cal mol(-1) K(-1) and -6.8 kcal mol(-1) at 25 °C, respectively; the interaction is hydrophobic in nature. The interaction of βLG with curcumin does not affect either the conformation or the state of association of βLG. Competitive ligand binding measurements, binding studies with denatured βLG, effect of pH on the curcumin-βLG interaction, Förster energy transfer measurements, and molecular docking studies suggest that curcumin binds to the central calyx of βLG. These binding studies have prompted the preparation and encapsulation of curcumin in βLG nanoparticles. Nanoparticles of βLG prepared by desolvation are found to encapsulate curcumin with >96% efficiency. The solubility of curcumin in βLG nanoparticle is significantly enhanced to ∼625 μM in comparison with its aqueous solubility (30 nM). Nanoparticles of βLG, by virtue of their ability to enhance solubility and stability of curcumin, may fit the choice as a carrier molecule.

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Aspergillus oryzae MTCC 5341, when grown on wheat bran as substrate, produces several extracellular acid proteases. Production of the major acid protease (constituting 34% of the total) by solid-state fermentation is optimized. Optimum operating conditions obtained are determined as pH 5, temperature of incubation of 30 degrees C, defatted soy flour addition of 4%, and fermentation time of 120 h, resulting in acid protease production of 8.64 x 10(5) U/g bran. Response-surface methodology is used to generate a predictive model of the combined effects of independent variables such as, pH, temperature, defatted soy flour addition, and fermentation time. The statistical design indicates that all four independent variables have significant effects on acid protease production. Optimum factor levels are pH 5.4, incubation temperature of 31 degrees C, 4.4% defatted soy flour addition, and fermentation time of 123 h to yield a maximum activity of 8.93 x 10(5) U/g bran. Evaluation experiments, carried out to verify the predictions, reveal that A. oryzae produces 8.47 x 10(5) U/g bran, which corresponds to 94.8% of the predicted value. This is the highest acid protease activity reported so far, wherein the fungus produces four times higher activity than previously reported [J Bacteriol 130(1): 48-56, 1977].

Thermal inactivation of glucose oxidase (GOD; ␤-Dglucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme, NaCl, and K 2 SO 4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60°C. The activation energy increased from 60.3 kcal mol ؊1 to 72.9, 76.1, and 88.3 kcal mol ؊1 , whereas the entropy of activation increased from 104 to 141, 147, and 184 cal⅐mol ؊1 ⅐deg ؊1 in the presence of 7.1 ؋ 10 ؊5 M lysozyme, 1 M NaCl, and 0.2 M K 2 SO 4 , respectively. The thermal unfolding of GOD in the temperature range of 25-90°C was studied using circular dichroism measurements at 222, 274, and 375 nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59°C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62°C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K 2 SO 4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.