Kinetic characterization of galacto-oligosaccharide (GOS) synthesis by three commercially important ß-galactosidases.

Many ß-galactosidases show large differences in galacto-oligosaccharide (GOS) production and lactose hydrolysis. In this study, a kinetic model is developed in which the effect of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of various ß-galactosidases is quantified. The use of oNPG as a competing substrate to lactose yields more information than can be obtained by examining only the conversion of lactose itself. The reaction rate with lactose or oligosaccharides as substrate relative to that with water as acceptor is much higher for the ß-galactosidase of Bacillus circulans than the bgalactosidases of Aspergillus oryzae and Kluyveromyces lactis. In addition, the ß-galactosidase of B.circulans has a high reaction rate with galactose as acceptor, in contrast to those of A. oryzae and K. lactis. The latter two are strongly inhibited by galactose. These differences explain why ß-galactosidase of B. circulans gives higher yields in GOS production than other ß-galactosidases. Many of the reaction rate constants for the ß-galactosidase isoforms of B. circulans increase with increasing molecular weight of the isoform. This indicates that the largest isoform ß-gal-A is most active in GOS production. However, its hydrolysis rate is also much higher than that of the other isoforms, which results in a faster hydrolysis of oligosaccharides as well.

ß-galactosidase stability at high substrate concentrations.

Enzymatic synthesis of galacto-oligosaccharides is usually performed at high initial substrate concentrations since higher yields are obtained. We report here on the stability of ß-galactosidase from Bacillus circulans at 25, 40, and 60°C in buffer, and in systems with initially 5.0 and 30% (w/w) lactose. In buffer, the half-life time was 220 h and 13 h at 25 and 40°C, respectively, whereas the enzyme was completely inactivated after two hours at 60°C. In systems with 5.0 and 30% (w/w) lactose, a mechanistic model was used to correct the oNPG converting activity for the presence of lactose, glucose, galactose, and oligosaccharides in the activity assay. Without correction, the stability at 5.0% (w/w) lactose was overestimated, while the stability at 30% (w/w) lactose was underestimated. The inactivation constant k d was strongly dependent on temperature in buffer, whereas only a slight increase in k d was found with temperature at high substrate concentrations. The enzyme stability was found to increase strongly with the initial substrate concentrations. The inactivation energy E a appeared to be lower at high initial substrate concentrations.

Effects of carbohydrates on the oNPG converting activity of ß-galactosidases.

The effects of high concentrations of carbohydrates on the o-nitrophenyl ß-d-galactopyranoside (oNPG) converting activity of ß-galactosidase from Bacillus circulans are studied to get a better understanding of the enzyme behavior in concentrated and complicated systems in which enzymatic synthesis of galacto-oligosaccharides is usually performed. The components that were tested were glucose, galactose, lactose, sucrose, trehalose, raffinose, Vivinal GOS, dextran-6000, dextran-70,000, and sarcosine. Small carbohydrates act as acceptors in the reaction. This speeds up the limiting step, which is binding of the galactose residue with the acceptor and release of the product. Simultaneously, both inert and reacting additives seem to cause some molecular crowding, which results in a higher enzyme affinity for the substrate. The effect of molecular crowding on the enzyme activity is small compared to the effect of carbohydrates acting in the reactions as acceptors. The effects of reactants on ß-galactosidases from B. circulans, A. oryzae, and K. lactis are compared.

Characterization of ß-galactosidase isoforms from Bacillus circulans and their contribution to GOS production.

A ß-galactosidase preparation from Bacillus circulans consists of four isoforms called ß-gal-A, ß-gal-B, ß-gal-C, and ß-gal-D. These isoforms differ in lactose hydrolysis and galacto-oligosaccharide (GOS) synthesis at low substrate concentrations. For this reason, using a selection of the isoforms may be relevant for GOS production, which is typically done at high substrate concentrations. At initial lactose concentrations in between 0.44 % and 0.68 % (w/w), ß-gal-A showed the least oligosaccharide formation, followed by ß-gal-B and ß-gal-C; most oligosaccharides were formed by ß-gal-D. The differences in behavior were confirmed by studying the thermodynamics of lactose conversion with isothermal titration calorimetry since especially ß-gal-A showed a different profile than the other isoforms. Also during the conversion of allolactose and 4-galactosyllactose at 0.44 % and 0.61 % (w/w), respectively, ß-gal-A and ß-gal-D showed clear differences. In contrast to above findings, the selectivity of the isoforms did hardly differ at an initial lactose concentration of 30 % (w/w), except for a slightly higher production of galactose with ß-gal-A. These differences were hypothesized to be related to the different accessibility of the active sites of the isoforms for different-sized reactants. The initial GOS formation rates of the isoforms indicate that ß-gal-A and ß-gal-B are the best isoforms for GOS production at high lactose concentrations.

Dynamic interfacial tension measurements with microfluidic Y-junctions.

Emulsification in microdevices (microfluidic emulsification) involves micrometer-sized droplets and fast interface expansion rates. In addition, droplets are formed in less than milliseconds, and therefore traditional tensiometric techniques cannot be used to quantify the actual interfacial tension. In this paper, monodisperse droplets formed at flat microfluidic Y-junctions were used to quantify the apparent dynamic interfacial tension during (microfluidic) emulsification. Hexadecane droplets were formed in ethanol-water solutions with a range of static interfacial tensions to derive a calibration curve, which was subsequently used to access the dynamic interfacial tension of hexadecane droplets formed in surfactant solutions. For SDS and Synperonic PEF108, various continuous- and disperse-phase (hexadecane) flow rates were studied, and these conditions were linked to interfacial tension effects, which also allowed convective transport of surfactants to be investiagted. On the basis of these findings, various strategies for the formation of emulsion droplets can be followed and are discussed.