Andrés Illanes

Synthesis of galacto-oligosaccharides by β-galactosidase from Aspergillus oryzae using partially dissolved and supersaturated solution of lactose

The effect of enzyme to substrate ratio, initial lactose concentration and temperature has been studied for the kinetically controlled reaction of lactose transgalactosylation with Aspergillus oryzae β-galactosidase, to produce prebiotic galacto-oligosaccharides (GOS). Enzyme to substrate ratio had no significant effect on maximum yield and specific productivity. Galacto-oligosaccharide syntheses at very high lactose concentrations (40, 50 and 60%, w/w, lactose monohydrate) were evaluated at different temperatures (40, 47.5 and 55°C). Within these ranges, lactose could be found as a supersaturated solution or a heterogeneous system with precipitated lactose, resulting in significant effect on GOS synthesis. An increase in initial lactose concentration produced a slight increase in maximum yield as long as lactose remained dissolved. Increase in temperature produced a slight decrease in maximum yield and an increase in specific productivity when supersaturation of lactose occurred during reaction. Highest yield of 29 g GOS/100 g lactose added was obtained at a lactose monohydrate initial concentration of 50% (w/w) and 47.5°C. Highest specific productivity of 0.38 g GOSh(-1) mg enzyme(-1) was obtained at lactose monohydrate initial concentration of 40% (w/w) and 55°C, where a maximum yield of 27 g GOS/100 g lactose added was reached. This reflects the complex interplay between temperature and initial lactose concentration on the reaction of synthesis. When lactose precipitation occurred, values of yields and specific productivities lower than 22 g GOS/100 g lactose added and 0.03 gGOSh(-1) mg enzyme(-1) were obtained, respectively.

Heterofunctional Hydrophilic–Hydrophobic Porous Silica as Support for Multipoint Covalent Immobilization of Lipases: Application to Lactulose Palmitate Synthesis

Lipase-catalyzed synthesis of sugar esters, as lactulose palmitate, requires harsh conditions, making it necessary to immobilize the enzyme. Therefore, a study was conducted to evaluate the effect of different chemical surfaces of hierarchical meso-macroporous silica in the immobilization of two lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), which exhibit esterase activity. Porosity and chemical surface of silica supports, before and after functionalization and after immobilization, were characterized by gas adsorption and Fourier transform infrared (FTIR) spectroscopy. PsL and AsL were immobilized in octyl (OS), glyoxyl (GS), and octyl-glyoxyl silica (OGS). Hydrolytic activity, thermal and solvent stability, and sugar ester synthesis were evaluated with those catalysts. The best support in terms of expressed activity was OS in the case of PsL (100 IU g(-1)), while OS and OGS were the best for AsL with quite similar expressed activities (60 and 58 IU g(-1), respectively). At 60 °C in aqueous media the more stable biocatalysts were GS-PsL and OGS-AsL (half-lives of 566 and 248 h, respectively), showing the advantage of a heterofunctional support in the latter case. Lactulose palmitate synthesis was carried out in acetone medium (with 4% of equilibrium moisture) at 40 °C obtaining palmitic acid conversions higher than 20% for all biocatalysts, being the highest of those obtained with OGS-AsL and OS-PsL. Therefore, screening of different chemical surfaces on porous silica used as supports for lipase immobilization allowed obtaining active and stable biocatalyst to be employed in the novel synthesis of lactulose palmitate.

Determination of the transgalactosylation activity of Aspergillus oryzae β-galactosidase: effect of pH, temperature, and galactose and glucose concentrations

The catalytic potential of β-galactosidase is usually determined by its hydrolytic activity over natural or synthetic substrates. However, this method poorly predicts enzyme behavior when transglycosylation instead of hydrolysis is being performed. A system for determining the transgalactosylation activity of β-galactosidase from Aspergillus oryzae was developed, and its activity was determined under conditions for the synthesis of galacto-oligosaccharides and lactulose. Transgalactosylation activity increased with temperature up to 55°C while the effect of pH was mild in the range from pH 2.5 to 5.5, decreasing at higher values. The effect of glucose and galactose on transgalactosylation activity was also assessed both in the reactions for the synthesis of galacto-oligosaccharides and lactulose and also in the reaction of hydrolysis of o-nitrophenyl β-d-galactopiranoside. Galactose was a competitive inhibitor and its effect was stronger in the reactions of transgalactosylation than in the reaction of hydrolysis. Glucose was a mild activator of β-galactosidase in the reaction of hydrolysis, but its mechanism of action was more complex in the reactions of transgalactosylation, having this positive effect only at low concentrations while acting as an inhibitor at high concentrations. This information is relevant to properly assess the effect of monosaccharides during the reactions of the synthesis of lactose-derived oligosaccharides, such as galacto-oligosaccharides and lactulose.

Galacto-oligosaccharides as protective molecules in the preservation of Lactobacillus delbrueckii subsp. bulgaricus.

In this work, the protective capacity of galacto-oligosaccharides in the preservation of Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 was evaluated. Lactobacillus bulgaricus was freeze-dried or dried over silica gel in the presence of three commercial products containing galacto-oligosaccharides. The freeze-dried samples were stored at 5 and 25°C for different periods of time. After desiccation, freeze-drying or storage, samples were rehydrated and bacterial plate counts were determined. According to the results obtained, all galacto-oligosaccharides assays demonstrated to be highly efficient in the preservation of L. bulgaricus. The higher content of galacto-oligosaccharides in the commercial products was correlated with their higher protective capacity. Galacto-oligosaccharides are widely known by their prebiotic properties. However, their role as protective molecules have not been reported nor properly explored up to now. In this work the protective capacity of galacto-oligosaccharides in the preservation of L. bulgaricus, a strain particularly sensitive to any preservation process, was demonstrated. The novel role of galacto-oligosaccharides as protective molecules opens up several perspectives in regard to their applications. The supplementation of probiotics with galacto-oligosaccharides allows the production of self-protected synbiotic products, galacto-oligosaccharides exerting both a prebiotic and protecting effect.

A pseudo steady‐state model for galacto‐oligosaccharides synthesis with β‐galactosidase from Aspergillus oryzae

A pseudo steady‐state model for the kinetically controlled synthesis of galacto‐oligosaccharides (GOS) with Aspergillus oryzae β‐galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of galactose, glucose, di, tri, tetra, and penta‐oligosaccharides during the synthesis, being able to describe the total GOS content in the reaction medium at the experimental conditions evaluated. Experimental results show that the formation of GOS containing only galactose residues is significant at high conversions of substrate, which was taken into account in the model. The formation of enzyme transition complexes was considered and reasonable assumptions were made to reduce the number of parameters to be determined. The model developed has 8 parameters; 2 of them were experimentally determined and the other 6 were estimated by fitting to the experimental data using multiresponse regression. Temperature effect on kinetic and affinity constants was determined in the range from 40 to 55°C, and the data were fitted to Arrhenius type equation. Parameters of the proposed model are independent from the enzyme load in the reaction medium and, differently from previously reported models, they have a clear biochemical meaning. The magnitude of the kinetic and affinity constants of the enzyme suggests that the liberation of galactose from the galactosyl–enzyme complex is a very slow reaction and such complex is driven into GOS formation. It also suggests that the affinity for sugars of the galactosyl–enzyme complex is higher than that of the free enzyme. Biotechnol. Bioeng. 2011;108: 2270–2279.

Transgalactosylation and hydrolytic activities of commercial preparations of β-galactosidase for the synthesis of prebiotic carbohydrates.

β-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: β-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of β-galactosidases could be tuned by changing the concentration of galactose donor.

Improvement of efficiency in the enzymatic synthesis of lactulose palmitate.

Sugar esters are considered as surfactants due to its amphiphilic balance that can lower the surface tension in oil/water mixtures. Enzymatic syntheses of these compounds are interesting both from economic and environmental considerations. A study was carried out to evaluate the effect of four solvents, temperature, substrate molar ratio, biocatalyst source, and immobilization methodology on the yield and specific productivity of lactulose palmitate monoester synthesis. Lipases from Pseudomonas stutzeri (PsL) and Alcaligenes sp. (AsL), immobilized in porous silica functionalized with octyl groups (adsorption immobilization, OS) and with glyoxyl-octyl groups (both adsorption and covalent immobilization, OGS), were used. The highest lactulose palmitate yields were obtained at 47 °C in acetone, for all biocatalysts, while the best lactulose:palmitic acid molar ratio differed according to the immobilization methodology, being 1:1 for AsL-OGS biocatalyst (20.7 ± 3%) and 1:3 for the others (30-50%).

Use of whey permeate containing in situ synthesised galacto-oligosaccharides for the growth and preservation of Lactobacillus plantarum

Galacto-oligosaccharides (GOS) are prebiotics that have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. GOS are commonly produced from lactose in an enzymatic reaction catalysed by β-galactosidase, named transglycosylation. Lactose is the main constituent of whey permeate (WP), normally wasted output from the cheese industry. Therefore, the main goal of this work was to optimise the synthesis of GOS in WP using β-galatosidase from Aspergillus oryzaea. WP and whey permeate enzymatically treated (WP-GOS) were used as culture media of Lactobacillus plantarum 299v. Lb. plantarum 299v attained the stationary phase in approximately 16 h, reaching 3·6 and 4·1×108 CFU/ml in WP and WP-GOS, respectively. The in situ synthesised GOS were not consumed during growth. No significant differences were observed in the growth kinetics of microorganisms in both media. After fermentation, microorganisms were dehydrated by freeze-drying and spray-drying and stored. The recovery of microorganisms after fermentation, dehydration and storage at 4 °C for at least 120 d was above 108 CFU/g. These studies demonstrated that WP is an appropriate substrate for the synthesis of GOS and the obtained product is also adequate as culture medium of Lb. plantarum 299v. The coexistence of GOS and dehydrated viable probiotic microorganisms, prepared using an effluent as raw material, represents the main achievement of this work, with potential impact in the development of functional foods.

Optimisation of synthesis of oligosaccharides derived from lactulose (fructosyl-galacto-oligosaccharides) with β-galactosidases of different origin.

Batch synthesis of fructosyl-galacto-oligosaccharides from lactulose was performed with commercial β-galactosidase preparations from Aspergillus oryzae, Kluyveromyces lactis and Bacillus circulans. The enzyme from A. oryzae produced the highest yield and specific productivity of synthesis, being selected for further studies. Optimization of fructosyl-galacto-oligosaccharides synthesis was carried out using response surface methodology, considering temperature and initial sugar concentration as variables and yield and specific productivity as response parameters. Maximum yield of 0.41 g g(-1) fructosyl-galacto-oligosaccharides was obtained at 70°C and 60% w/w lactulose concentration, while maximum specific productivity of 1.2 g h(-1)mg(-1) was obtained at 70°C and 40% w/w lactulose concentration.

Repeated-batch operation for the synthesis of lactulose with β-galactosidase immobilized by aggregation and crosslinking

Synthesis of lactulose under repeated-batch operation was done with cross-linked aggregates of Aspergillus oryzae β-galactosidase (CLAGs). The effect of the crosslinking agent to enzyme mass ratio and cross-linking time were first evaluated. Best results were obtained at 5.5gdeglutaraldehyde/g enzyme at 5h of cross-linking, obtaining a specific activity of 15,000IUg(-1), with 30% immobilization yield. CLAG was more stable than the free enzyme under non-reactive conditions with a half-life of 123h at 50°C and when operated in repeated-batch mode, yield and productivity was 3.8 and 4.3 times higher. Maximum number of batches was determined considering biocatalyst replacement at 50% residual activity. 98 and 27 batches could be performed under such criterion at fructose/lactose molar ratio of 4 and 20 respectively, reflecting that enzyme stability is strongly affected by the sugars distribution in the reaction medium.