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The effects of water activity (aw), pH, and temperature on transglycosylation activity of α-L-fucosidase from Thermotoga maritima in the synthesis of fucosylated oligosaccharides were evaluated using different water-organic cosolvent reaction systems. The optimum conditions of transglycosylation reaction were the pH range between 7 and 10 and temperature 90–95 °C. The addition of organic cosolvent decreased α-L-fucosidase transglycosylation activity in the following order: acetone > dimethyl sulfoxide (DMSO) > acetonitrile (0.51 > 0.42 > 0.18 mM/h). However, the presence of DMSO and acetone enhanced enzyme-catalyzed transglycosylation over hydrolysis as demonstrated by the obtained transglycosylation/hydrolysis rate (rT/H) values of 1.21 and 1.43, respectively. The lowest rT/H was calculated for acetonitrile (0.59), though all cosolvents tested improved the transglycosylation rate in comparison to a control assay (0.39). Overall, the study allowed the production of fucosylated oligosaccharides in water-organic cosolvent reaction media using α-L-fucosidase from T. maritima as biocatalyst. [ABSTRACT FROM AUTHOR]

In Mexico, whey is discarded into drains without treatment, which represents an environmental problem. It is known that whey is a mixture of nutrients that increase biochemical demand (BOD) and chemical oxygen demand (COD), exceeding the limits established by national and international standards. Some uses of whey have been proposed to reduce the negative impact it produces on the environment. Some of these uses are the production of high‐value raw materials, separation of macromolecules of industrial interest, and the generation of energy, among others. The integral use of waste under a biorefinery scheme represents a viable alternative for the use of this waste. In this way, the chemical composition of the waste can be used to recover compounds with high added value. The main objective of this review is to propose a dairy whey biorefinery to use components of commercial interest. This biorefinery process involves three stages: the first is the separation of lactoferrin using a polyacrylamide matrix (PAM) impregnated with copper; the second is lactic fermentation using the residue resulting from the separation of lactoferrin, from which lactic acid, hydrolyzed proteins, and probiotics will be obtained. Finally, in the third stage, the residue from the previous stage can be used to generate biogas. © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd [ABSTRACT FROM AUTHOR]

Abstract: An exopolysaccharide (EPS) producing strain of Streptococcus thermophilus was evaluated for the production of Panela cheese using two total solids milk (TSM) concentrations (12.5 and 17.5g/100mL). This ropy strain increased cheese yield; nevertheless, with 12.5 TSM the increment was higher than with 17.5 TSM. Analysis of cheese composition showed that with 12.5 TSM, the ropy strain increased moisture, but did not change the fat or non fat solids on dry weight basis (dwb), suggesting that the increment of the yield is only due to water retention. In 17.5 TSM cheeses the ropy strain caused an increase in the moisture and fat (dwb), suggesting that besides water retention, fat also contributed to the yield. The difference in yield increment could be explained by cheese composition: higher fat content creates a more hydrophobic environment, which would expel more water than the cheese with lower fat content. Electron microscopy showed EPS attached to the protein matrix of the cheeses. In 17.5 TSM cheeses EPS was observed around the milk fat globules (MFG), confirming that higher TSM causes EPS to bind the MFG besides binding the protein matrix, retaining fat within the cheese. Sensory evaluation demonstrated that ropy cheeses were softer and creamier. [Copyright &y& Elsevier]

Abstract: The activity of Kluyveromyces lactis β-galactosidase increased when it was measured in the presence of either β-lactoglobulin or bovine serum albumin, whereas α-lactalbumin showed no effect on enzyme activity. Heating β-lactoglobulin further increased β-galactosidase activity. Affinity chromatography assays demonstrated that lactase bound specifically to β-lactoglobulin, resulting in enzyme activation. Heating β-lactoglobulin in the presence of lactose resulted in a reaction between the protein and the sugar (lactolation), which diminished the binding capacity of the protein to the enzyme and therefore its activating effect. It was concluded that β-lactoglobulin raises lactase activity through two different mechanisms, one of which depends on the release of sulfhydryl groups by heat treatment from the denatured protein, and the other a result of the ability of the native protein to bind the enzyme. [Copyright &y& Elsevier]

Native beta-lactoglobulin binds and increases the activity of Kluyveromyces lactis beta-galactosidase. Construction of a three-dimensional (3D) model of beta-lactoglobulin showed that lysine residues 15, 47, 69, and 138 are the most exposed ones, thus the ones more likely to interact with beta-galactosidase. Molecular docking estimated the interaction energies of amino acid residues with either lactose or succinic anhydride, showing that Lys(138) is the most likely to react with both. Affinity chromatography demonstrated that succinylated beta-lactoglobulin diminished its ability to bind to the enzyme. Furthermore, when activity was measured in the presence of succinylated beta-lactoglobulin, its activating effect was lost. Since succinylation specifically blocks Lys epsilon-amino groups, their loss very likely causes the disappearance of the activating effect. Results show that the activating effect of beta-lactoglobulin on beta-galactosidase activity is due to the interaction between both proteins and that this interaction is very likely to occur through the Lys epsilon-amino groups of beta-lactoglobulin.

The present study evaluated the influence of water activity and lactose concentration on the synthesis of galactooligosaccharides (GOS), by means of a hyperthermophilic beta-glycosidase in an organic system. The production of GOS gradually grew as water activity increased in the reaction system; later, their synthesis decreased as water activity increased. The authors used the response surface methodology to study how different water activities and different concentrations of lactose influenced the synthesis of GOS and their length. In every case, the variable that proved to have the greatest effect on GOS synthesis was water activity. Maximum GOS3 synthesis was reached at a water activity interval of 0.44-0.57, with lactose concentrations of 0.06%-0.1%, while GOS4 and GOS5 maxima were reached at water activity intervals of 0.47-0.57 and 0.49-0.60, respectively. The research showed that higher water activity was required to synthesize GOS of greater length. Synthesis of GOS would then depend on the flexibility of the enzyme, which in turn would depend on water activity of the reaction system. This hypothesis was supported by experiments in which the reaction temperature was modified in order to change the flexibility of the enzyme, thus leading to longer GOS.

The secondary structure of Kluyveromyces lactis beta-galactosidase was determined by circular dichroism. It is mainly a beta-type protein, having 22% beta-turns, 14% parallel beta-sheet, 25% antiparallel beta-sheet, 34% unordered structure, and only 5% alpha-helix. The structure-activity relationship as a function of the pH was also studied. The pH conditions leading to the highest secondary structure content (100% ellipticity) of the enzyme was found at pH 7.0; at pH 6.5-7.0, the percent ellipticity decreased slightly, suggesting little structural change, but the activity decreased significantly, probably because of variations in critical residues. On the other hand, at pH's above 7.0, a more noticeable change in ellipticity was observed due to structural changes; the CD analysis showed a small increase in the helical content toward higher pH, whereas the maximum activity was found at pH 7.5, meaning that the changes produced in the secondary structure at this pH favored the interaction between the enzyme and the substrate.