Immobilization of the ß-galactosidase enzyme by encapsulation in polymeric matrices for application in the dairy industry.

Lactose intolerance affects approximately 65% of the global adult population, leading to the demand for lactose-free products. The enzyme ß-galactosidase (ßG) is commonly used in the industry to produce such products, but its recovery after lactose hydrolysis is challenging. In this scenario, the study aims to encapsulate ßG within capsules, varying in dimensions and wall materials, to ensure their suitability for efficient industrial recovery. The enzyme ßG was encapsulated through ionic gelation using alginate and its blends with pectin, maltodextrin, starch, or whey protein as wall materials. The capsules produced underwent evaluation for encapsulation efficiency, release profiles, activity of the ßG enzyme, and the decline in enzyme activity when reused over multiple cycles. Alginate at 5% wt/vol concentrations, alone or combined with polymers such as maltodextrin, starch, or whey protein, achieved encapsulation efficiencies of approximately 98%, 98%, 80%, and 88%, respectively. The corresponding enzyme recovery rates were 34%, 19%, 31%, and 48%. Capsules made with an alginate-pectin blend exhibited no significant hydrolysis and maintained an encapsulation efficiency of 79%. Encapsulation with alginate alone demonstrated on poor retention of enzyme activity, showing a loss of 74% after just 4 cycles of reuse. Conversely, when alginate was mixed with starch or whey protein concentrate, the loss of enzyme activity was less than 40% after 4 reuses. These results highlight the benefits of combining encapsulation materials to improve enzyme recovery and reuse, offering potential economic advantages for the dairy industry.

Probiotic and functional potential of lactic acid bacteria isolated from pulque and evaluation of their safety for food applications.

Pulque is a traditional Mexican non-distilled alcoholic beverage to which several beneficial functions are attributed, mainly associated with gastrointestinal health, which can be explained by the presence of probiotic bacteria in its microbiota. Therefore, the objective of this work was to evaluate the safety, probiotic activity, and functional characteristics of seven strains of lactic acid bacteria (LAB) isolated from pulque using the probiotic strain Lactobacillus acidophilus NCFM as control. The LAB isolates were identified by 16S rRNA sequencing and MALDI Biotyper® MS as belonging to three different Lactobacillaceae genera and species: Lactiplantibacillus plantarum, Levilactobacillus brevis and Lacticaseibacillus paracasei. Most strains showed resistance to gastric juice, intestinal juice and lysozyme (10 mg/L). In addition, all strains exhibited bile salt hydrolase (BSH) activity and antibacterial activity against the pathogenic strain Listeria monocytogenes. Additionally, cell surface characteristics of LAB were evaluated, with most strains showing good hydrophobicity, auto-aggregation, and co-aggregation towards enteropathogenic Escherichia coli and L. monocytogenes. In terms of safety, most of the strains were sensitive to the tested antibiotics and only the Lact. paracasei UTMB4 strain amplified a gene related to antibiotic resistance (mecA). The strains Lact. plantarum RVG2 and Lact. plantarum UTMB1 presented γ-hemolytic activity, and the presence of the virulence-related gene agg was identified only in UTMB1 strain. Regarding functional characterization, the tested bacteria showed good ß-galactosidase activity, antioxidant activity and cholesterol reduction Based on principal component analysis (PCA) and heat mapping, and considering the strain Lact. acidophilus NCFM as the probiotic reference, the strains Lacticaseibacillus paracasei UTMB4, Lactiplantibacillus plantarum RVG4 and Levilactobacillus brevis UTMB2 were selected as the most promising probiotic strains. The results of this study highlighted the probiotic, functional and safety traits of LAB strains isolated from pulque thus supporting the health benefits attributed to this ancestral beverage.

Application of Lactose-Free Whey Protein to Greek Yogurts: Potential Health Benefits and Impact on Rheological Aspects and Sensory Attributes.

The application of ß-galactosidase in the fermentation of milk enables the acquirement of lower levels of lactose that are tolerated by lactose maldigesters and can reduce the nutritional consequences of avoiding dairy products. The present study evaluated the viability of the fortification of lactose-free prebiotic Greek yogurt formulas with whey protein concentrate (WPC). Two rotational central composite designs (RCCDs) were applied: one to perform the hydrolysis of the whey protein concentrate and another for the yogurt formulations (α = 2 with 2 central points and 4 axial points). Two ß-galactosidase enzymes obtained from Kluyveromyces lactis were used. The content of lactose, glucose, galactose, and lactic acid were determined in the WPC, milk (pasteurized and powdered), and yogurts. The three best formulations regarding the attributes' viscosity, syneresis, firmness, and elasticity were sensorially evaluated by using a nine-point hedonic scale. A microbiological analysis was performed after 48 h of yogurt production. The characterization of the products and the comparison of the results obtained were evaluated using the Student's T test and the analysis of variance with Tukey's test (p-values < 0.05). The application of a lactose-free WPC promoted viscosity, firmness, and elasticity. The syneresis was reduced, and whey increased the protein and calcium content. Lactose-free WPC can be used as a partial substitute for skimmed powdered milk in yogurts. The obtained results are encouraging with respect to the production of lactose-free Greek yogurts by the dairy industry.

Galactooligosaccharides: Physiological benefits, production strategies, and industrial application.

The concern for better life quality has been encouraging the bioprocess industries to develop technological strategies to obtain new biomolecules. Galactooligosaccharides (GOS) are an important class of food-grade oligosaccharides, being classified as non-digestible, and which present prebiotic potential, promoting better conditions of health and well-being. The main benefits include the selective stimulation of beneficial microorganisms, the decrease in the formation of toxic compounds, the increase in the absorption of minerals, improvement of an immune response, and a reduction in the severity of obesity and diabetes. This review approaches the recent methodologies and strategies to obtain GOS, their health benefits, purification, and technological properties for industrial application. Improvements in the process are continuously being investigated, with the technique of enzyme immobilization representing a potentially promising strategy. Sustainable GOS productions have been reported by the use of agro-industrial residues, such as cheese whey. Despite these advances, the main concern of the process consists in the low yield, which implies high investments in the purification of the bioproducts. Technological and nutritional approaches to the GOS application in different industrial sectors are also reported.

His-tag ß-galactosidase supramolecular performance.

ß-Galactosidase is an important biotechnological enzyme used in the dairy industry, pharmacology and in molecular biology. In our laboratory we have overexpressed a recombinant ß-galactosidase in Escherichia coli (E. coli). This enzyme differs from its native version (ß-GalWT) in that 6 histidine residues have been added to the carboxyl terminus in the primary sequence (ß-GalHis), which allows its purification by immobilized metal affinity chromatography (IMAC). In this work we compared the functionality and structure of both proteins and evaluated their catalytic behavior on the kinetics of lactose hydrolysis. We observed a significant reduction in the enzymatic activity of ß-GalHis with respect to ß-GalWT. Although, both enzymes showed a similar catalytic profile as a function of temperature, ß-GalHis presented a higher resistance to the thermal inactivation compared to ß-GalWT. At room temperature, ß-GalHis showed a fluorescence spectrum compatible with a partially unstructured protein, however, it exhibited a lower tendency to the thermal-induced unfolding with respect to ß-GalWT. The distinctively supramolecular arranges of the proteins would explain the effect of the presence of His-tag on the enzymatic activity and thermal stability.

Artificial intelligence applied to enzymatic hydrolysis of lactose: improving the control of industrial processes / Inteligência artificial aplicada a hidrólise enzimática da lactose: melhorando o controle dos processos industriais

Lactose is the main carbohydrate in milk, and its absorption occurs via enzymatic hydrolysis, generating glucose and galactose. Lactose intolerance is the reduction of intestinal hydrolysis capacity due to hypolactasia, which results in the need to consume dairy foods with low levels of this carbohydrate. ß-galactosidase enzymes are used in dairy industries to hydrolyze lactose, thereby allowing intolerant consumers access to dairy products without the negative health implications. Alternative and official analytical methods are used to quantify the carbohydrate content resulting from enzymatic hydrolysis. The objective of this study was to evaluate the enzymatic hydrolysis of two distinct industrial enzymes produced by the microorganisms Bacillus licheniformis and Kluyveromyces lactis using three analytical methods: enzymatic method, cryoscopy, and high performance liquid chromatography (HPLC) using artificial intelligence to improve the control of the industrial processes. After adding the enzymes to skim milk, time kinetics was performed by collecting samples at time 0, every 10 min for 1 h, and every 30 min until the end of 5 h of hydrolysis. In 97% of the cases, a decrease in lactose concentration was observed by HPLC, followed by the deepening of the cryoscopic point. Glucose measurements by absorbance and HPLC quantification were correlated (r = 0.79; p < 0.01) but not concordant (p < 0.01). It was concluded that by means of artificial intelligence, it was possible to indirectly estimate lactose concentration using an algorithm that associates cryoscopy and glucose concentration.(AU)

O principal carboidrato do leite é a lactose e a sua absorção ocorre devido à hidrólise enzimática, gerando glicose e galactose. A intolerância à lactose é a redução da capacidade de hidrólise intestinal devido à hipolactasia, gerando a necessidade do consumo de alimentos lácteos com baixo teor deste carboidrato. As enzimas ß-galactosidase são utilizadas nas indústrias de laticínios para hidrolisar a lactose, proporcionando ao consumidor intolerante a possibilidade de ingerir os produtos lácteos sem prejuízos à saúde. Para quantificar o conteúdo de carboidratos resultante da hidrólise enzimática, são utilizados métodos analíticos alternativos e oficiais. O objetivo deste estudo foi avaliar a hidrólise enzimática de duas enzimas industriais distintas produzidas pelos microrganismos Bacillus licheniformis e Kluyveromyces lactis, por meio de três métodos analíticos: método enzimático, crioscopia e HPLC. A inteligência artificial foi utilizada para melhorar o controle dos processos industriais. Após a adição das enzimas ao leite desnatado, foi realizada a cinética de tempo coletando as amostras no tempo 0, a cada 10 minutos, até completar 1 hora de reação, e a cada 30 minutos até serem atingidas 5 horas de reação de hidrólise. Em 97% dos casos, a diminuição da concentração de lactose por HPLC acompanhou o aprofundamento do ponto crioscópico. As medições de glicose por absorbância e HPLC foram correlacionadas (r = 0,79; p < 0,01), mas não concordantes (p < 0,01). Concluiu-se que, por meio da inteligência artificial, é possível estimar indiretamente a concentração de lactose a partir de um algoritmo que associa a crioscopia e a concentração de glicose.(AU)

Trends in lactose-derived bioactives: synthesis and purification.

Lactose obtained from cheese whey is a low value commodity despite its great potential as raw material for the production of bioactive compounds. Among them, prebiotics stand out as valuable ingredients to be added to food matrices to build up functional foods, which currently represent the most active sector within the food industry. Functional foods market has been growing steadily in the recent decades along with the increasing awareness of the World population about healthy nutrition, and this is having a strong impact on lactose-derived bioactives. Most of them are produced by enzyme biocatalysis because of molecular precision and environmental sustainability considerations. The current status and outlook of the production of lactose-derived bioactive compounds is presented with special emphasis on downstream operations which are critical because of the rather modest lactose conversion and product yields that are attainable. Even though some of these products have already an established market, there are still several challenges referring to the need of developing better catalysts and more cost-effective downstream operations for delivering high quality products at affordable prices. This technological push is expected to broaden the spectrum of lactose-derived bioactive compounds to be produced at industrial scale in the near future.

Complementary experimental/docking approach for determining chitosan and carboxymethylchitosan ability for the formation of active polymer-ß-galactosidase adducts.

The spontaneous aggregation of chitosan and carboxymethylchitosan polymers can be advantageous for the enzyme confinement on these colloidal systems during immobilization processes. The initial crucial step involves the polymer-enzyme adduct formation. The objective here is to determine the interactions that drive the adduct formation between these polymers and ß-galactosidase from Bacillus circulans. The chemical characterization of chitosan and its carboxymethyl-derivate allowed to explain their colloidal behavior and design the four-unit fragments ligands used for the docking study. The deacetylation degree (0.6 times lower), isoelectric point (5.2 instead 6.4) and substitution degree (DSO = 1.779 and DS2N = 0.441) of carboxymenthylchitosan are due to the hydroxide concentration (>25%) and 30 °C modification conditions. Favorable Van der Waals and H-bond interactions between chitosan-ß-galactosidase and contribution of electrostatic attraction mediated by calcium ions for carboxymethylchitosan-ß-galactosidase explained the zeta potential and dynamic light scattering results at pH 7.0. These interactions occur onto the external surface of this galactosidase, without affecting the catalytic activity. A cross-linked enzyme aggregates-type model was proposed for the formation of the adducts, based on the complementary experimental-docking results. They contribute understanding the behavior of polyelectrolyte chitosan-derived matrices for enzyme immobilization.

Enzymatic production of lactulose by fed-batch and repeated fed-batch reactor.

The effects of the most significant operational variables on reactor performance of fed-batch and repeated fed-batch were evaluated in the lactulose production by enzymatic transgalactosylation. Feed flowrate in the fed stage (F) and fructose to lactose molar ratio (Fr/L) were the variables that mostly affected the values ​​of lactulose yield (YLu), lactulose productivity (πLu) and selectivity of transgalactosylation (SLu/TOS). Maximum YLu of 0.21 g lactulose per g lactose was obtained at 50% w/w inlet carbohydrates concentration (IC) of, 50 °C, Fr/L 8, F 1 mL⋅min-1, 200 IU∙gLactose-1 reactor enzyme load and pH 4.5. At these conditions the selectivity was 7.4, productivity was 0.71 gLu∙g-1∙h-1and lactose conversion was 0.66. The operation by repeated fed batch increases the efficiency of use of the biocatalysts (EB) and the accumulated productivity compared to batch and fed batch operation with the same biocatalyst. EB obtained was 4.13 gLu∙mgbiocatalyst protein-1, 10.6 times higher than in fed-batch.

Effect of product partition on the synthesis of butyl-ß-D-galactoside from Aspergillus oryzae.

The effect of donor substrate and products partitioning on the performance of butyl-ß-galactoside synthesis with Aspergillus oryzae ß-galactosidase was studied. Firstly, the partition coefficient of the donor substrate (lactose) and the reaction products (glucose, galactose and butyl-ß-galactoside) were determined in the aqueous and organic phases of the reaction medium. In the temperature range studied (30 to 50 °C), butyl ß-galactoside was roughly 130 and 30-fold more soluble in the organic phase than lactose and the monosaccharides, respectively. Afterward, the effect of the 1-butanol/ aqueous phase ratio (α) on the reaction was evaluated in the range from 0.25 to 4. Results show that higher values of α reduce the incidence of secondary hydrolysis by favoring the extraction of butyl-ß-galactoside into the organic phase where it is not hydrolyzed, leading to higher yields. Also, major interfacial properties for butyl-ß-galactoside were determined at 25 °C.

Development of a Hybrid Bioinorganic Nanobiocatalyst: Remarkable Impact of the Immobilization Conditions on Activity and Stability of ß-Galactosidase.

Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from ß-galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected ß-galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications.

Synthesis of magnetic nanoparticles functionalized with histidine and nickel to immobilize His-tagged enzymes using ß-galactosidase as a model.

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using ß-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant ß-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized ß-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant ß-galactosidase, showing its potential application in other high-molecular-weight enzymes.

Immobilization of ß-galactosidase from Bacillus licheniformis for application in the dairy industry.

The food industry has developed a wide range of products with reduced lactose to allow people with intolerance to consume dairy products. Although ß-galactosidase has extensive applications in the food, pharma, and biotechnology industries, the enzymes are high-cost catalysts, and their use makes the process costly. Immobilization is a viable strategy for enzyme retention inside a reactor, allowing its reuse and application in continuous processes. Here, we studied the immobilization of ß-galactosidase from Bacillus licheniformis in ion exchange resin. A central composite rotational design (CCRD) was proposed to evaluate the immobilization process in relation to three immobilization solution variables: offered enzyme activity, ionic strength, and pH. The conditions that maximized the response were offered enzyme activity of 953 U, 40 mM ionic strength, and pH 4.0. Subsequently, experiments were performed to provide additional stabilization for biocatalyst, using a buffer solution pH 9.0 at 25 °C for 24 h, and crosslinking with different concentrations of glutaraldehyde. The stabilization step drastically impacted the activity of the immobilized enzyme, and the reticulation with different concentrations of glutaraldehyde showed significant influence on the activity of the immobilized enzyme. In spite of substantially affecting the initial activity of the immobilized enzyme, higher reagent concentrations (3.5 g L-1) were effective for maintaining stability related to the number of cycles of the enzyme immobilized. The ß-galactosidase from Bacillus licheniformis immobilized in Duolite A568 is a promising technique to produce reduced or lactose-free dairy products, as it allows reuse of the biocatalyst, decreasing operational costs.Key Points• Immobilization of ß-galactosidase from Bacillus licheniformis in batch reactor• Influence of buffer pH and ionic concentration and offered enzyme activity on immobilization• Influence of glutaraldehyde on operational stability.

Antarctic Rahnella inusitata: A Producer of Cold-Stable ß-Galactosidase Enzymes.

There has been a recent increase in the exploration of cold-active ß-galactosidases, as it offers new alternatives for the dairy industry, mainly in response to the current needs of lactose-intolerant consumers. Since extremophilic microbial compounds might have unique physical and chemical properties, this research aimed to study the capacity of Antarctic bacterial strains to produce cold-active ß-galactosidases. A screening revealed 81 out of 304 strains with ß-galactosidase activity. The strain Se8.10.12 showed the highest enzymatic activity. Morphological, biochemical, and molecular characterization based on whole-genome sequencing confirmed it as the first Rahnella inusitata isolate from the Antarctic, which retained 41-62% of its ß-galactosidase activity in the cold (4 °C-15 °C). Three ß-galactosidases genes were found in the R. inusitata genome, which belong to the glycoside hydrolase families GH2 (LacZ and EbgA) and GH42 (BglY). Based on molecular docking, some of these enzymes exhibited higher lactose predicted affinity than the commercial control enzyme from Aspergillus oryzae. Hence, this work reports a new Rahnella inusitata strain from the Antarctic continent as a prominent cold-active ß-galactosidase producer.

Production and partial purification by PEG/citrate ATPS of a ß-galactosidase from the new promising isolate Cladosporium tenuissimum URM 7803.

ß-Galactosidase production, partial purification and characterization by a new fungal were investigated. Partial purification was performed by aqueous two-phase system (ATPS) using polyethylene glycol (PEG) molar mass, PEG concentration, citrate concentration and pH as the independent variables. Purification factor (PF), partition coefficient (K) and yield (Y) were the responses. After identification by rDNA sequencing and classification as Cladosporium tenuissimum URM 7803, this isolate achieved a maximum cell concentration and ß-galactosidase activity of 0.48 g/L and 462.1 U/mL, respectively. ß-Galactosidase partitioned preferentially for bottom salt-rich phase likely due to hydrophobicity and volume exclusion effect caused in the top phase by the high PEG concentration and molar mass. The highest value of PF (12.94) was obtained using 24% (w/w) PEG 8000 g/mol and 15% (w/w) citrate, while that of Y (79.76%) using 20% (w/w) PEG 400 g/mol and 25% (w/w) citrate, both at pH 6. The enzyme exhibited optimum temperature in crude and ATPS extracts in the ranges 35-50 °C and 40-55 °C, respectively, and optimum pH in the range 3.0-4.5, with a fall of enzyme activity under alkaline conditions. Some metal ions and detergents inhibited, while others stimulated enzyme activity. Finally, C. tenuissimum URM 7803 ß-galactosidase showed a profile suitable for prebiotics production.

Recent advances in ß-galactosidase and fructosyltransferase immobilization technology.

The highly demanding conditions of industrial processes may lower the stability and affect the activity of enzymes used as biocatalysts. Enzyme immobilization emerged as an approach to promote stabilization and easy removal of enzymes for their reusability. The aim of this review is to go through the principal immobilization strategies addressed to achieve optimal industrial processes with special care on those reported for two types of enzymes: ß-galactosidases and fructosyltransferases. The main methods used to immobilize these two enzymes are adsorption, entrapment, covalent coupling and cross-linking or aggregation (no support is used), all of them having pros and cons. Regarding the support, it should be cost-effective, assure the reusability and an easy recovery of the enzyme, increasing its stability and durability. The discussion provided showed that the type of enzyme, its origin, its purity, together with the type of immobilization method and the support will affect the performance during the enzymatic synthesis. Enzymes' immobilization involves interdisciplinary knowledge including enzymology, nanotechnology, molecular dynamics, cellular physiology and process design. The increasing availability of facilities has opened a variety of possibilities to define strategies to optimize the activity and re-usability of ß-galactosidases and fructosyltransferases, but there is still great place for innovative developments.

Immobilization of Aspergillus oryzae ß-galactosidase in cation functionalized agarose matrix and its application in the synthesis of lactulose.

Aspergillus oryzae ß-galactosidase was immobilized in in-house quaternary ammonium agarose (QAA) and used for the first time in the synthesis of lactulose. A biocatalyst was obtained with a specific activity of 24,690 IUH∙g-1; protein immobilization yield of 97% and enzyme immobilization yield of 76% were obtained at 30 °C in 10 mM phosphate buffer pH 7 for standard size agarose at 100 mgprotein∙gsupport-1 which the maximum protein load of QAA. Highest yield and specific productivity of lactulose were 0.24 g∙g-1 and 9.78 g∙g-1 h-1 respectively, obtained at pH 6, 100 IUH∙g lactose-1 enzyme/lactose ratio and 12 lactose/fructose molar ratio. In repeated-batch operation with the immobilized enzyme, the cumulative mass of lactulose per unit mass of contacted protein and cumulative specific productivity were higher than obtained with the soluble enzyme since the first batch. After enzyme activity exhaustion, the enzyme was desorbed and QAA support was reused without alteration in its maximum enzyme load capacity and without detriment in yield, productivity and selectivity in the batch synthesis of lactulose with the resulting biocatalyst. This significantly decreases the economic impact of the support, presenting itself as a distinctive advantage of immobilization by ionic interaction.

ß-Galactosidases from a Sequence-Based Metagenome: Cloning, Expression, Purification and Characterization.

Stabilization ponds are a common treatment technology for wastewater generated by dairy industries. Large proportions of cheese whey are thrown into these ponds, creating an environmental problem because of the large volume produced and the high biological and chemical oxygen demands. Due to its composition, mainly lactose and proteins, it can be considered as a raw material for value-added products, through physicochemical or enzymatic treatments. ß-Galactosidases (EC 3.2.1.23) are lactose modifying enzymes that can transform lactose in free monomers, glucose and galactose, or galactooligosacharides. Here, the identification of novel genes encoding ß-galactosidases, identified via whole-genome shotgun sequencing of the metagenome of dairy industries stabilization ponds is reported. The genes were selected based on the conservation of catalytic domains, comparing against the CAZy database, and focusing on families with ß-galactosidases activity (GH1, GH2 and GH42). A total of 394 candidate genes were found, all belonging to bacterial species. From these candidates, 12 were selected to be cloned and expressed. A total of six enzymes were expressed, and five cleaved efficiently ortho-nitrophenyl-ß-galactoside and lactose. The activity levels of one of these novel ß-galactosidase was higher than other enzymes reported from functional metagenomics screening and higher than the only enzyme reported from sequence-based metagenomics. A group of novel mesophilic ß-galactosidases from diary stabilization ponds' metagenomes was successfully identified, cloned and expressed. These novel enzymes provide alternatives for the production of value-added products from dairy industries' by-products.

Synthesis of Butyl-ß-D-Galactoside in the Ternary System: Acetone/1-Butanol/Aqueous Solution.

The enzymatic synthesis of short-tailed alkyl glucosides is generally carried out in an aqueous-organic biphasic reaction medium with a rather low fatty alcohol concentration in the aqueous phase (where the synthesis occurs). Thus, hydrolytic reactions have a significant impact on the synthesis performance. Given this background, the use of acetone as cosolvent was studied for the synthesis of butyl-ß-galactoside with Aspergillus oryzae ß-galactosidase. The liquid-liquid equilibrium of the reaction mixture components (acetone/1-butanol/aqueous solution) was determined and the single- and two-phase regions were defined at 30, 40, and 50°C. It was observed that the liquid-liquid equilibrium of the ternary system acetone/1-butanol/water differs significantly from the one obtained using an aqueous solution (50 mM McIlvaine buffer pH 4.5; 5 g L-1) instead of water. This is mainly because of the salting-out effect of the buffer; nevertheless, the presence of lactose also altered the equilibrium. Having this in mind, the effects of temperature (30 and 50°C) and reaction mixture composition were assessed. Three general conditions were evaluated: single-phase ternary system (30% acetone), two-phase ternary system (10% acetone) and two-phase binary system (0% acetone). Acetone had a deleterious effect on enzyme stability at 50°C, leading to low reaction yields. However, no enzyme deactivation was detected at 30°C. Moreover, a reaction yield of 0.98 mol mol-1 was attained in the 30/50/20% (w/w) mixture of acetone/1-butanol/aqueous solution. This very high yield can be explained by the huge increase in the concentration of 1-butanol and the reduction of water activity. The synthesis was carried out using also the ß-galactosidase immobilized in glyoxal-agarose and amino-glyoxal-agarose, and by aggregation and crosslinking. In the case of agarose-derived catalysts, two average particle diameters were assessed to evaluate the presence of internal mass transfer limitations. Best yield (0.88 mol mol-1) was obtained with glyoxal-agarose derivatives and the particle size had non-effect on yield. The chemical structure of butyl-ß-galactoside was determined by NMR and FT-IR.

Synthesis of Lactulose in Continuous Stirred Tank Reactor With ß-Galactosidase of Apergillus oryzae Immobilized in Monofunctional Glyoxyl Agarose Support.

Lactulose synthesis from fructose and lactose in continuous stirred tank (CSTR) reactor operation with glyoxyl-agarose immobilized Aspergillus oryzae ß-galactosidase is reported for the first time. The effect of operational variables: inlet concentrations of sugar substrates, temperature, feed substrate molar ratio, enzyme loading and feed flow rate was studied on reactor performance. Even though the variation of each one affected to a certain extent lactulose yield (Y Lactulose ), specific productivity (π Lactulose ) and selectivity of the reaction (lactulose/transgalactosylated oligosaccharides molar ratio) (S Lu/TOS ), the most significant effects were obtained by varying the inlet concentrations of sugar substrates and the feed substrate molar ratio. Maximum Y Lactulose of 0.54 g⋅g-1 was obtained at 50°C, pH 4.5, 50% w/w inlet concentrations of sugar substrates, feed flowrate of 12 mL⋅min-1, fructose/lactose molar ratio of 8 and reactor enzyme load of 29.06 IU H ⋅mL-1. At such conditions S Lu/TOS was 3.7, lactose conversion (X Lactose ) was 0.39 and total transgalactosylation yield was 0.762 g⋅g-1, meaning that 76% of the reacted lactose corresponded to transgalactosylation and 24% to hydrolysis, which is a definite advantage of this mode of operation. Even though X Lactose in CSTR was lower than in other reported modes of operation for lactulose synthesis, transgalactosylation was more favored over hydrolysis which reduced the inhibitory effect of galactose on ß-galactosidase.