Stabilization of ß-D-galactosidase in solution containing chitosan-based membrane: Central composite rotatable design.

ß-D-galactosidase is a hydrolase enzyme capable of hydrolyzing lactose in milk-based foods. Its free form can be inactivated in solution during the production of low-dosage lactose foods. Then, it is important to study strategies for avoiding the free enzyme inactivation with the aim of circumventing this problem. The stabilization of ß-D-galactosidase in aqueous solution after interactions with chitosan/eucalyptus sawdust composite membrane proved to be a potential strategy when optimized by central composite rotatable (CCR) design. In this case, the best experimental conditions for ß-D-galactosidase partitioning and stability in an aqueous medium containing the chitosan-based composite membrane reinforced with eucalyptus sawdust were i) enzyme/buffer solution ratio of 0.0057, ii) pH 5.6, iii) membrane mass of 50 mg, and iv) temperature lower than 37 °C. Significance was found for the linear enzyme/buffer solution ratio, linear temperature, and quadratic pH (p < 0.05) in the interval between 0 and 60 min of study. In the interval between 60 and 120 min, there was significance (p < 0.12) for linear temperature, the temperature-enzyme/buffer solution ratio interaction and the interaction between linear pH and linear enzyme/buffer solution ratio. The Pareto charts and response surfaces clearly showed all the effects of the experimental variables on the stabilization of ß-D-galactosidase in solution after interactions with the chitosan composite membrane. In this case, industrial food reactors covered with chitosan/eucalyptus sawdust composite membrane could be a strategy for the hydrolysis of lactose during milk-producing processes.

Enzymes for production of whey protein hydrolysates and other value-added products.

Whey is a byproduct of dairy industries, the aqueous portion which separates from cheese during the coagulation of milk. It represents approximately 85-95% of milk's volume and retains much of its nutrients, including functional proteins and peptides, lipids, lactose, minerals, and vitamins. Due to its composition, mainly proteins and lactose, it can be considered a raw material for value-added products. Whey-derived products are often used to supplement food, as they have shown several physiological effects on the body. Whey protein hydrolysates are reported to have different activities, including antihypertensive, antioxidant, antithrombotic, opioid, antimicrobial, cytomodulatory, and immuno-modulatory. On the other hand, galactooligosaccharides obtained from lactose can be used as prebiotic for beneficial microorganisms for the human gastrointestinal tract. All these compounds can be obtained through physicochemical, microbial, or enzymatic treatments. Particularly, enzymatic processes have the advantage of being highly selective, more stable than chemical transformations, and less polluting, making that the global enzyme market grow at accelerated rates. The sources and different products associated with the most used enzymes are particularly highlighted in this review. Moreover, we discuss metagenomics as a tool to identify novel proteolytic enzymes, from both cultivable and uncultivable microorganisms, which are expected to have new interesting activities. Finally enzymes for the transformation of whey sugar are reviewed. In this sense, carbozymes with ß-galactosidase activity are capable of lactose hydrolysis, to obtain free monomers, and transgalactosylation for prebiotics production. KEY POINTS: • Whey can be used to obtain value-added products efficiently through enzymatic treatments • Proteases transform whey proteins into biopeptides with physiological activities • Lactose can be transformed into prebiotic compounds using ß-galactosidases.

In vitro and in vivo digestibility of prebiotic galactooligosacharides synthesized by ß-galactosidase from Lactobacillus delbruecki subsp. bulgaricus CRL450.

BACKGROUND: The general assumption that prebiotics reach the colon without any alterations has been challenged. Some in vitro and in vivo studies have demonstrated that 'non-digestible' oligosaccharides are digested to different degrees depending on their structural composition. In the present study, we compared different methods aiming to assess the digestibility of oligosaccharides synthesized by ß-galactosidase (ß-gal) of Lactobacillus delbruecki subsp. bulgaricus CRL450 (CRL450-ß-gal) from lactose, lactulose and lactitol. RESULTS: In the simulated gastrointestinal fluid method, no changes were observed. However, the oligosaccharides synthesized by CRL450-ß-gal were partially hydrolyzed in vitro, depending on their structure and composition, with rat small intestinal extract (RSIE) and small intestinal brush-border membrane vesicles (BBMV) from pig. Digestion of some oligosaccharides increased when mixtures were fed to C57BL/6 mice used as in vivo model; however, lactulose-oligosaccharides were the most resistant to the physiological conditions of mice. In general ß (1→6) linked products showed higher resistance compared to ß (1→3) oligosaccharides. CONCLUSION: In vitro digestion methods, without disaccharidases, may underestimate the importance of carbohydrates hydrolysis in the small intestine. Although BVMM and RSIE digestion assays are appropriate in vitro methods for these studies, in vivo studies remain the most reliable for understanding what actually happens in the digestion of oligosaccharides. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Overexpression of miR-22-3p and miR-29c-3p in CFU-Hill colonies is related to senescence process among children with low birth weight.

BACKGROUND: The current study mainly focused on provide further insights into the association of the miR-22-3p and miR-29c-3p expression in CFU-Hill colonies with birth weight and senescence process in children. METHODS: This cross-sectional study evaluated 61 children (32 boys, 29 girls). The CFU-Hill colonies number was evaluated in vitro by cell culture technique and senescence was detected by ß-galactosidase (SA-ß-Gal) assay. Expression of miR-22-3p and miR-29c-3p isolated from CFU-Hill colonies were detected using quantitative real-time polymerase chain reaction. RESULTS: Birth weight was correlated with both CFU-Hill colonies and %SA-ß-Gal positive staining. Multivariate linear regression analysis revealed that the senescence was a predictor of the lower CFU-Hill colonies number, while only the birth weight was a predictor of senescence of CFU-Hill colonies. Overexpression of miR-22-3p and miR-29c-3p was observed in CFU-Hill colonies isolated from children with low birth weight (LBW). Interestingly, we found a significant correlation between %SA-ß-Gal cells staining positive for both miR-22-3p and miR-29c-3p. CONCLUSION: The LBW is associated with decreased CFU-Hill colonies number and high senescence of these cells. The overexpression of miR-22-3p and miR-29c-3p may be partially responsible for this alteration due to regulation of several pathways related to the senescence process. IMPACT: The study establishes a significant correlation between birth weight and the number of CFU-Hill colonies, suggesting that birth weight could be a predictive biomarker for vascular health in children. Data indicates that cellular senescence is a predictor of reduced CFU-Hill colony numbers. This suggests that the aging process of these cells could be an important factor in understanding the vascular health issues in children with low birth weight. The overexpression of miR-22-3p and miR-29c-3p in children with low birth weight and their correlation with increased cellular senescence highlight these microRNAs as possible molecular mechanisms influencing the aging of CFU-Hill colonies.

Oxidative stress, cardiomyocytes premature senescence and contractile dysfunction in in vitro and in vivo experimental models of Chagas disease.

AIMS: The relationship between redox imbalance and cardiovascular senescence in infectious myocarditis is unknown. Thus, the aim of this study was to investigate whether cardiomyocytes parasitism, oxidative stress and contractile dysfunction can be correlated to senescence-associated ß-galactosidase (SA-ß-Gal) activity in Trypanosoma cruzi-infection in vitro and in vivo. METHODS: Uninfected, T. cruzi-infected untreated and benznidazole (BZN)-treated H9c2 cardiomyocytes and rats were investigated. Parasitological, prooxidant, antioxidant, microstructural, and senescence-associated markers were quantified in vitro and in vivo. RESULTS: T. cruzi infection triggered intense cardiomyocytes parasitism in vitro and in vivo, which was accompanied by reactive oxygen species (ROS) upregulation, lipids, proteins and DNA oxidation in cardiomyocytes and cardiac tissue. Oxidative stress was parallel to microstructural cell damage (e.g., increased cardiac toponin I levels) and contractile dysfunction in cardiomyocytes in vitro and in vivo, whose severity accompanied a premature cellular senescence-like phenotype revealed by increased senescence-associated ß-galactosidase (SA-ß-Gal) activity and DNA oxidation (8-OHdG). Cellular parasitism (e.g., infection rate and parasite load), myocarditis and T. cruzi-induced prooxidant responses were attenuated by early BZN administration to interrupt the progression of T. cruzi infection, protecting against SA-ß-gal-based premature cellular senescence, microstructural damage and contractile deterioration in cardiomyocytes from T. cruzi-infected animals. CONCLUSION: Our findings indicated that cell parasitism, redox imbalance and contractile dysfunction were correlated to SA-ß-Gal-based cardiomyocytes premature senescence in acute T. cruzi infection. Therefore, in addition to controlling parasitism, inflammation and oxidative stress; inhibiting cardiomyocytes premature senescence should be further investigated as an additional target of specific Chagas disease therapeutics.

Synthesis of thiodisaccharides related to 4-thiolactose. Specific structural modifications increase the inhibitory activity against E. coli ß-galactosidase.

In the search for new glycosidase inhibitors, a set of benzyl ß-D-Gal-S-(1→4)-3-deoxy-4-thio-α-D-hexopyranosides was synthesized. Diverse configurations were installed at C-2 and C-4 of the glucose residue. The benzyl glycosidic group was kept intact or substituted by an electron-donating or electron-withdrawing group that could also participate in hydrogen bonding. All thiodisaccharides were found to be inhibitors of E. coli ß-galactosidase. In general, benzyl thiodisaccharides were better inhibitors than those substituted (NO2 or NH2) on the benzyl ring. Thiodisaccharides containing a hexopyranoside, instead of a pentopyranoside, showed a weaker inhibitory activity, except for those having the α-D-xylo configuration, which exhibited inhibition constants of the same order of magnitude. These and previous results indicated that the inhibition process by thiodisaccharides is strongly dependent on the configuration of the 3-deoxy-4-thiopyranoside, as well as its substitution pattern (such as the presence of a benzyl glycoside). The enzyme-inhibitor interaction during the hydrolysis process involves a conformational selection resulting from rotation around the thioglycosidic bond and the flexibility of the terminal six-membered ring. Thus, the mentioned structural features of the inhibitor could give rise to favorable ground state conformations for the interaction with the enzyme, similar to those found for selected thiodisaccharides in the bound state. These studies demonstrated that the performance of thiodisaccharides as enzyme inhibitors could be increased by selecting the appropriate configuration and substitution of the hexopyranoside replacing the glucose moiety of 4-thiolactose.

Higher Oxidative Stress in Endometriotic Lesions Upregulates Senescence-Associated p16ink4a and ß-Galactosidase in Stromal Cells.

Endometriosis affects a significant proportion of women worldwide; however, no definitive cure for this disease has been discovered to date. Oxidative stress promotes endometriotic lesion maintenance in the peritoneal cavity in women. Furthermore, there is evidence of the mitogen-activated protein kinase (MAPK) signaling pathway and senescence involvement in the physiopathogenesis of endometriosis. Reactive oxygen species (ROS) cause oxidative damage and are expected to trigger senescence in the endometrium while also causing alterations in MAPK signaling. However, the role of ROS in the senescence-associated phenotype in endometriosis remains unknown. In this context, this study attempted to delineate the pathways linking ROS to senescence in endometrial and endometriotic lesions of healthy individuals and those with endometriosis. Our results indicate a higher presence of ROS in endometriotic lesions, and the upregulation of MAPK. Furthermore, we show that endometriotic lesions in stromal cells stimulated with hydrogen peroxide develop more senescence traits than eutopic and non-endometriosis endometrium. Overall, endometriotic cells respond differently to extracellular distress. Our contribution to further research in this field contributed to the roadmap of endometriosis' search for alternative treatments.

Lack of ZNF365 Drives Senescence and Exacerbates Experimental Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant activation of the alveolar epithelium, the expansion of the fibroblast population, and the accumulation of extracellular matrix. Global gene expression of human lung fibroblasts stimulated with TGFß-1, a strong fibrotic mediator revealed the overexpression of ZNF365, a zinc finger protein implicated in cell cycle control and telomere stabilization. We evaluated the expression and localization of ZNF365 in IPF lungs and in the fibrotic response induced by bleomycin in WT and deficient mice of the orthologous gene Zfp365. In IPF, ZNF365 was overexpressed and localized in fibroblasts/myofibroblasts and alveolar epithelium. Bleomycin-induced lung fibrosis showed an upregulation of Zfp365 localized in lung epithelium and stromal cell populations. Zfp365 KO mice developed a significantly higher fibrotic response compared with WT mice by morphology and hydroxyproline content. Silencing ZNF365 in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and increased senescence markers, including Senescence Associated ß Galactosidase activity, p53, p21, and the histone variant γH2AX. Our findings demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and suggest a protective role since its absence increases experimental lung fibrosis mechanistically associated with the induction of cell senescence.

Development of a biocomposite based on alginate/gelatin crosslinked with genipin for ß-galactosidase immobilization: Performance and characteristics.

In this work, we studied the development of a biocomposite formulated with alginate and gelatin, crosslinked with genipin for application as support for ß-galactosidase immobilization. Also, the biocomposites with the immobilized enzyme were characterized by thermal analyses and SAXS (size, density, and interconnectivity of alginate rods) for a detailed analysis of the microstructure, as well as the thermal and operational stabilities of the enzyme. The structural modifications of the biocomposite determined by SAXS demonstrate that the addition of both genipin and enzyme produced a significant reduction in size and density of the Ca(II)-alginate rods. Immobilized ß-galactosidase could be stored for 175 days under refrigeration maintaining 80% of its initial activity. Moreover, 90% of its relative activity was kept after 11 reuses in a batch process of lactose hydrolysis. Thus, the biocomposite proved to be effective as support for enzyme immobilization.

One-step purification of a recombinant beta-galactosidase using magnetic cellulose as a support: Rapid immobilization and high thermal stability.

For the first time, this work reported the one-step purification and targeted immobilization process of a ß-galactosidase (Gal) with the Cellulose Binding Domain (CBD) tag, by binding it to different magnetic cellulose supports. The process efficiency after ß-galactosidase-CBD immobilization on magnetic cellulose-based supports showed values of approximately 90% for all evaluated enzymatic loads. Compared with free Gal, derivatives showed affinity values between ß-galactosidase and the substrate 1.2 × higher in the lactose hydrolysis of milk. ß-Galactosidase-CBD's oriented immobilization process on supports increased the thermal stability of the immobilized enzyme by up to 7 × . After 15 cycles of reuse, both enzyme preparations showed a relative hydrolysis percentage of 50% of lactose in milk. The oriented immobilization process developed for purifying recombinant proteins containing the CBD tag enabled the execution of both steps simultaneously and quickly and the obtention of ß-galactosidases with promising catalytic characteristics for application in the food and pharmaceutical industries.

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.

ß-Galactosidase from Kluyveromyces lactis: Characterization, production, immobilization and applications - A review.

A review on the enzyme ß-galactosidase from Kluyveromyces lactis is presented, from the perspective of its structure and mechanisms of action, the main catalyzed reactions, the key factors influencing its activity, and selectivity, as well as the main techniques used for improving the biocatalyst functionality. Particular attention was given to the discussion of hydrolysis, transglycosylation, and galactosylation reactions, which are commonly mediated by this enzyme. In addition, the products generated from these processes were highlighted. Finally, biocatalyst improvement techniques are also discussed, such as enzyme immobilization and protein engineering. On these topics, the most recent immobilization strategies are presented, emphasizing processes that not only allow the recovery of the biocatalyst but also deliver enzymes that show better resistance to high temperatures, chemicals, and inhibitors. In addition, genetic engineering techniques to improve the catalytic properties of the ß-galactosidases were reported. This review gathers information to allow the development of biocatalysts based on the ß-galactosidase enzyme from K. lactis, aiming to improve existing bioprocesses or develop new ones.

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.

Challenges and perspectives of the ß-galactosidase enzyme.

The enzyme ß-galactosidase has great potential for application in the food and pharmaceutical industries due to its ability to perform the hydrolysis of lactose, a disaccharide present in milk and in dairy by-products. It can be used in free form, in batch processes, or in immobilized form, which allows continuous operation and provides greater enzymatic stability. The choice of method and support for enzyme immobilization is essential, as the performance of the biocatalyst is strongly influenced by the properties of the material used and by the interaction mechanisms between support and enzyme. Therefore, this review showed the main enzyme immobilization techniques, and the most used supports for the constitution of biocatalysts. Also, materials with the potential for immobilization of ß-galactosidases and the importance of their biotechnological application are presented. KEY POINTS: • The main methods of immobilization are physical adsorption, covalent bonding, and crosslinking. • The structural conditions of the supports are determining factors in the performance of the biocatalysts. • Enzymatic hydrolysis plays an important role in the biotechnology industry.

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.

High performance biocatalyst based on ß-d-galactosidase immobilized on mesoporous silica/titania/chitosan material.

A new support for the immobilization of ß-d-galactosidase from Kluyveromyces lactis was developed, consisting of mesoporous silica/titania with a chitosan coating. This support presents a high available surface area and adequate pore size for optimizing the immobilization efficiency of the enzyme and, furthermore, maintaining its activity. The obtained supported biocatalyst was applied in enzyme hydrolytic activity tests with o-NPG, showing high activity 1223 Ug-1, excellent efficiency (74%), and activity recovery (54%). Tests of lactose hydrolysis in a continuous flow reactor showed that during 14 days operation, the biocatalyst maintained full enzymatic activity. In a batch system, after 15 cycles, it retained approximately 90% of its initial catalytic activity and attained full conversion of the lactose 100% (±12%). Additionally, with the use of the mesoporous silica/titania support, the biocatalyst presented no deformation and fragmentation, in both systems, demonstrating high operational stability and appropriate properties for applications in food manufacturing.

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.

ß-galactosidase from Kluyveromyces lactis in genipin-activated chitosan: An investigation on immobilization, stability, and application in diluted UHT milk.

The objective of this research was to evaluate the immobilization of the enzyme ß-galactosidase in a genipin-activated chitosan support. The influence of the number of spheres and substrate concentration on immobilization yield (IY) and enzyme activity (EA) was analyzed using experimental design. Thermal, operational and storage stabilities were assessed, and the enzymatic derivatives were characterized by thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The TGA showed that the enzymatic derivatives kept their thermal behavior, and the SEM images revealed smooth surfaces in all the spheres. The optimized conditions for the immobilization process were 4.57 mg·mL-1 of spheres and a substrate concentration of 10 mM (IY = 84.13%; EA = 24.97 U·g-1). Thermal stability was enhanced at 10 and 37 °C, enabling four successive cycles of lactose hydrolysis in diluted UHT milk. Therefore, the immobilized enzyme in genipin-activated chitosan has potential for lactose hydrolysis and applications in the food industry.

Synthesis of b(1 ? 3) and b(1 ? 6) galactooligosaccharides from lactose and whey using a recombinant b-galactosidase from Pantoea anthophila

BACKGROUND: Milk whey, a byproduct of the dairy industry has a negative environmental impact, can be used as a raw material for added-value compounds such as galactooligosaccharides (GOS) synthesis by bgalactosidases. RESULTS: B-gal42 from Pantoea anthophila strain isolated from tejuino belonging to the glycosyl hydrolase family GH42, was overexpressed in Escherichia coli and used for GOS synthesis from lactose or milk whey. Crude cell-free enzyme extracts exhibited high stability; they were employed for GOS synthesis reactions. In reactions with 400 g/L lactose, the maximum GOS yield was 40% (w/w) measured by HPAEC-PAD, corresponding to 86% of conversion. This enzyme had a strong predilection to form GOS with b(1 ? 6) and b (1 ? 3) galactosyl linkages. Comparing GOS synthesis between milk whey and pure lactose, both of them at 300 g/L, these two substrates gave rise to a yield of 38% (60% of lactose conversion) with the same product profile determined by HPAEC-PAD. CONCLUSIONS: B-gal42 can be used on whey (a cheap lactose source) to produce added value products such as galactooligosaccharides.