The paper introduces ultrasonic technology for real-time, nondestructive, precision monitoring of enzyme-catalyzed reactions in solutions and in complex opaque media. The capabilities of the technology are examined in a comprehensive analysis of the effects of a variety of diverse factors on the performance of enzyme β-galactosidase in formulations for reduction of levels of lactose in infant milks. These formulations are added to infant's milk bottles prior to feeding to overcome the frequently observed intolerance to lactose (a milk sugar), a serious issue in healthy development of infants. The results highlight important impediments in the development of these formulations and also illustrate the capability of the described ultrasonic tools in the assessment of the performance of enzymes in complex reaction media and in various environmental conditions.

Abstract:The rapidly growing field of chemical catalysis is dependent on analytical methods for non-destructive real-time monitoring of chemical reactions in complex systems such as emulsions, suspensions and gels, where most analytical techniques are limited in their applicability, especially if the media is opaque, or if the reactants/products do not possess optical activity. High-resolution ultrasonic spectroscopy is one of the novel technologies based on measurements of parameters of ultrasonic waves propagating through analyzed samples, which can be utilized for real-time non-invasive monitoring of chemical reactions. It does not require optical transparency, optical markers and is applicable for monitoring of reactions in continuous media and in micro/nano bioreactors (e.g., nanodroplets of microemulsions). The technology enables measurements of concentrations of substrates and products over the whole course of reaction, analysis of time profiles of the degree of polymerization and molar mass of polymers and oligomers, evolutions of reaction rates, evaluation of kinetic mechanisms, measurements of kinetic and equilibrium constants and reaction Gibbs energy. It also provides tools for assessments of various aspects of performance of catalysts/enzymes including inhibition effects, reversible and irreversible thermal deactivation. In addition, ultrasonic scattering effects in dispersions allow real-time monitoring of structural changes in the medium accompanying chemical reactions.

A low-temperature mini-emulsion medium for the enzymatic resolution of 1-phenylethanol is described for the first time. The enzymatic hydrolysis resolution of 1-phenylethyl esters with different chain-lengths in the presence of Candida antarctica lipase B in mini-emulsion media was shown to be significantly controlled by temperature. In this system, the direct effect of temperature on the mini-emulsion size was observed. For the longer 1-phenylethyl ester, 1-phenylethyl dodecanoate, the enzymatic resolution was promoted exclusively at low temperatures. The preparative mini-emulsion enzymatic reaction of 1-phenylethyl dodecanoate at 4°C afforded the isolation of (R)-phenylethanol with a yield of 36 % with an ee of 99 %. (S)-Phenylethanol was isolated with a 51 % yield with an ee of 79 %.