Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT‐stabilized water‐in‐oil microemulsions: I. Effect of pH and water content on reaction kinetics

Abstract

Lipolase and Lipozyme are produced in large quantities (as a result of genetic engineering and overexpression) for the detergents market and provide a cheap source of highly active biocatalysts. Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RmL) have been isolated in partially purified form from commercial preparations of Lipolase and Lipozyme, respectively. These lipases were solubilized in Aerosol-OT (AOT)-stabilized water-in-oil (w/o) microemulsions in n-heptane. HIL and RmL activity in these microemulsions was assayed by spectrophotometric measurement of the initial rate of p-nitophenyl butyrate hydrolysis, and by chromatographic determination of the initial rate of octyl decanoate synthesis from 1-octanol and decanoic acid. The hydrolytic activity of HIL in microemulsions measured as a function of buffer pH prior to dispersal, followed a sigmoidal profile with the highest activities observed at alkaline pHs. This broadly matches the pH-activity profile for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. The hydrolytic activity of RmL in the same microemulsions, measured as a function of pH, gave a bell-shaped profile with a maximum activity at pH 7.5. Again, the observed pH-activity profile was similar to that reported for a purified RmL in a tributyrin-based aqueous emulsion assay. In contrast, the esterification activity exhibited by both HIL and RmL in AOT microemulsions over the available range pH 6.1 to 10.4, decreases as the pH increases, most likely reflecting the effect of substrate ionization. The dependence of the hydrolytic and condensation activity of HIL on R, the mole ratio of water to surfactant, were similar with both profiles exhibiting a maximum at R = 5. The hydrolytic and esterification activities of RmL followed similar R-dependent profiles, but the profiles in this case exhibited a maximum at R = 10. The water activities at these R values were directly measured as 0.78 and 0.9, respectively. Measured water activities were unperturbed by the presence of lipase at the concentrations used in these studies.

Enzymatic Assay Method for Evaluating the Lipase Activity in Complex Extracts from Transgenic Corn Seed

“…Similar characteristics were observed for Humicola lanuginose lipase and Rhizomucor miehei lipase (16). Our preliminary experiments showed a good reproducibility of this method when a commercial CVL was used.…”

“…Crooks et al (16) described an aqueous system in which a substrate p-nitrophenyl butyrate was first dissolved in acetone and then mixed in a buffer. The method, however, had a low reproducibility possibly due to a poorly defined surface area of oil (acetone) droplets in the absence of surfactants.…”

Production of margarine fats by enzymatic interesterification with silica‐granulated Thermomyces lanuginosa lipase in a large‐scale study

“…A highly hydrophobic carrier enhanced lipase activity for alcoholysis reactions, whereas the reverse effect was observed for acylglycerol synthesis (7). Comparison of hydrolysis and esterification behavior of T. lanuginosa and R. miehei lipases was also carried out in AOT-stabilized water-in-oil microemulsions (2,3). For the immobilized T. lanuginosa lipase on resin, the optimal temperature was 10°C higher than that of the free lipase.…”

“…Previously T. lanuginosa lipase was investigated mostly in solvent systems in free-state or immobilized forms (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). From kinetic studies, it was found that immobilized T. lanuginosa lipase had different characteristics depending on the solvent systems and the hydrophobicity of carriers (2,7).…”

“…Lipozyme TL IM has sn-1,3 specificity and hydrophilic character. It is less expensive than the commonly applied commercial lipase Lipozyme IM (Rhizomucor miehei lipase) and offers an opportunity for industry to reduce the process cost and make the production of low-price plastic fats economically competitive to the conventional chemical randomization.Previously T. lanuginosa lipase was investigated mostly in solvent systems in free-state or immobilized forms (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). From kinetic studies, it was found that immobilized T. lanuginosa lipase had different characteristics depending on the solvent systems and the hydrophobicity of carriers (2,7).…”