LacdiNAc synthase B4GALNT3 has a unique PA14 domain and suppresses N-glycan capping.

Structural variation of N-glycans is essential for the regulation of glycoprotein functions. GalNAcß1-4GlcNAc (LacdiNAc or LDN), a unique subterminal glycan structure synthesized by B4GALNT3 or B4GALNT4, is involved in the clearance of N-glycoproteins from the blood and maintenance of cell stemness. Such regulation of glycoprotein functions by LDN is largely different from that by the dominant subterminal structure, N-acetyllactosamine (Galß1-4GlcNAc, LacNAc). However, the mechanisms by which B4GALNT activity is regulated and how LDN plays different roles from LacNAc remain unclear. Here, we found that B4GALNT3 and four have unique domain organization containing a noncatalytic PA14 domain, which is a putative glycan-binding module. A mutant lacking this domain dramatically decreases the activity toward various substrates, such as N-glycan, O-GalNAc glycan, and glycoproteins, indicating that this domain is essential for enzyme activity and forms part of the catalytic region. In addition, to clarify the mechanism underlying the functional differences between LDN and LacNAc, we examined the effects of LDN on the maturation of N-glycans, focusing on the related glycosyltransferases upstream and downstream of B4GALNT. We revealed that, unlike LacNAc synthesis, prior formation of bisecting GlcNAc in N-glycan almost completely inhibits LDN synthesis by B4GALNT3. Moreover, the presence of LDN negatively impacted the actions of many glycosyltransferases for terminal modifications, including sialylation, fucosylation, and human natural killer-1 synthesis. These findings demonstrate that LDN has significant impacts on N-glycan maturation in a completely different way from LacNAc, which could contribute to obtaining a comprehensive overview of the system regulating complex N-glycan biosynthesis.

A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways.

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.

Efficacy and Findings of a Blinded Randomized Reintroduction Phase for the Low FODMAP Diet in Irritable Bowel Syndrome.

BACKGROUND & AIMS: The efficacy of a low fermentable oligo-, di-, monosaccharides and polyols (FODMAP) diet in irritable bowel syndrome (IBS) is well established. After the elimination period, a reintroduction phase aims to identify triggers. We studied the impact of a blinded reintroduction using FODMAP powders to objectively identify triggers and evaluated the effect on symptoms, quality of life, and psychosocial comorbidities. METHODS: Responders to a 6-week low FODMAP diet, defined by a drop in IBS symptom severity score (IBS-SSS) compared with baseline, entered a 9-week blinded randomized reintroduction phase with 6 FODMAP powders (fructans, fructose, galacto-oligosaccharides, lactose, mannitol, sorbitol) or control (glucose). A rise in IBS-SSS (≥50 points) defined a FODMAP trigger. Patients completed daily symptom diaries and questionnaires for quality of life and psychosocial comorbidities. RESULTS: In 117 recruited patients with IBS, IBS-SSS improved significantly after the elimination period compared with baseline (150 ± 116 vs 301 ± 97, P < .0001, 80% responders). Symptom recurrence was triggered in 85% of the FODMAP powders, by an average of 2.5 ± 2 FODMAPs/patient. The most prevalent triggers were fructans (56%) and mannitol (54%), followed by galacto-oligosaccharides, lactose, fructose, sorbitol, and glucose (respectively 35%, 28%, 27%, 23%, and 26%) with a significant increase in abdominal pain at day 1 for sorbitol/mannitol, day 2 for fructans/galacto-oligosaccharides, and day 3 for lactose. CONCLUSION: We confirmed the significant benefit of the low FODMAP diet in tertiary-care IBS. A blinded reintroduction revealed a personalized pattern of symptom recurrence, with fructans and mannitol as the most prevalent, and allows the most objective identification of individual FODMAP triggers. Ethical commission University hospital of Leuven reference number: s63629; Clinicaltrials.gov number: NCT04373304.

Hepatoprotective activity of Lactéol® forte and quercetin dihydrate against thioacetamide-induced hepatic cirrhosis in male albino rats.

Liver cirrhosis is a silent disease in humans and is experimentally induced by many drugs and toxins as thioacetamide (TAA) in particular, which is the typical model for experimental induction of hepatic fibrosis. Thus, the objective of the present study was to elucidate the possible protective effects of lactéol® forte (LF) and quercetin dihydrate (QD) against TAA-induced hepatic damage in male albino rats. Induction of hepatotoxicity was performed by TAA injection (200 mg/kg I/P, twice/ week) in rats. LF (1 × 109 CFU/rat 5 times/week) and QD (50 mg/kg 5 times/week) treated groups were administered concurrently with TAA injection (200 mg/kg I/P, twice/ week). The experimental treatments were conducted for 12 weeks. Hepatotoxicity was evaluated biochemically by measuring alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) in the serum and histopathologically with the scoring of histopathological changes besides histochemical assessment of collagen by Masson's trichrome and immunohistochemical analysis for α-smooth muscle actin (α-SMA), Ki67 and caspase-3 expression in liver sections. Our results indicated that LF and QD attenuated some biochemical changes and histochemical markers in TAA-mediated hepatotoxicity in rats by amelioration of biochemical markers and collagen, α-SMA, Ki67 and caspase3 Immunoexpression. Additionally, LF and QD supplementation downregulated the proliferative, necrotic, fibroblastic changes, eosinophilic intranuclear inclusions, hyaline globules and Mallory-like bodies that were detected histopathologically in the TAA group. In conclusion, LF showed better hepatic protection than QD against TAA-induced hepatotoxicity in rats by inhibiting inflammatory reactions with the improvement of some serum hepatic transaminases, histopathological picture and immunohistochemical markers.

Lactobacillus amylovorus Promotes Lactose Utilization in Small Intestine and Enhances Intestinal Barrier Function in Intrauterine Growth Restricted Piglets.

BACKGROUND: Intrauterine growth restriction (IUGR) resulted in high mortality and many physiological defects of piglets, causing huge economic loss in the swine industry. Lactobacillus amylovorus (L. amylovorus) was identified as one of the main differential bacteria between IUGR and normal piglets. However, the effects of L. amylovorus on the growth performance and intestinal health in IUGR piglets remained unclear. OBJECTIVES: This study aimed to investigate the promoting effects of L. amylovorus Mafic1501, a new strain isolated from normal piglets, on the growth performance and intestinal barrier functions in IUGR piglets. METHODS: Newborn mice or piglets were assigned into 3 groups: CON (normal birth weight, control), IUGR (low birth weight), and IUGR+L. amy (low birth weight), administered with sterile saline or L. amylovorus Mafic1501, respectively. Growth performance, lactose content in the digesta, intestinal lactose transporter, and barrier function parameters were profiled. IPEC-J2 cells were cultured to verify the effects of L. amylovorus Mafic1501 on lactose utilization and intestinal barrier functions. RESULTS: L. amylovorus Mafic1501 elevated body weight and average daily gain of IUGR mice and piglets (P < 0.05). The lactose content in the ileum was decreased, whereas gene expression of glucose transporter 2 (GLUT2) was increased by L. amylovorus Mafic1501 in IUGR piglets during suckling period (P < 0.05). Besides, L. amylovorus Mafic1501 promoted intestinal barrier functions by increasing the villus height and relative gene expressions of tight junctions (P < 0.05). L. amylovorus Mafic1501 and its culture supernatant decreased the lactose level in the medium and upregulated gene expressions of transporter GLUT2 and tight junction protein Claudin-1 of IPEC-J2 cells (P < 0.05). CONCLUSION: L. amylovorus Mafic1501 improved the growth performance of IUGR piglets by promoting the lactose utilization in small intestine and enhancing intestinal barrier functions. Our results provided the new evidence of L. amylovorus Mafic1501 for its application in the swine industry.

A FRET based ultrasensitive fluorescent aptasensor for 6'-sialyllactose detection.

As a kind of human milk oligosaccharide, 6'-sialyllactose (6'-SL) plays an important role in promoting infant brain development and improving infant immunity. The content of 6'-SL in infant formula milk powder is thus one of the important nutritional indexes. Since the lacking of efficient and rapid detection methods for 6'-SL, it is of great significance to develop specific recognition elements and establish fast and sensitive detection methods for 6'-SL. Herein, using 6'-SL specific aptamer as the recognition element, catalytic hairpin assembly as the signal amplification technology and quantum dots as the signal label, a fluorescence biosensor based on fluorescence resonance energy transfer (FRET) was constructed for ultra-sensitive detection of 6'-SL. The detection limit of this FRET-based fluorescent biosensor is 0.3 nM, and it has some outstanding characteristics such as high signal-to-noise ratio, low time-consuming, simplicity and high efficiency in the actual sample detection. Therefore, it has broad application prospect in 6'-SL detection.

Inhaled JAK Inhibitor GDC-0214 Nanoaggregate Powder Exhibits Improved Pharmacokinetic Profile in Rats Compared to the Micronized Form: Benefits of Thin Film Freezing.

Asthma is a common chronic disease affecting the airways in the lungs. The receptors of allergic cytokines, including interleukin (IL)-4, IL-5, and IL-13, trigger the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, which involves the pathogenesis of asthma. GDC-0214 is a JAK inhibitor that was developed as a potent and selective target for the treatment of asthma, specifically targeting the lungs. While inhaled GDC-0214 is a promising novel treatment option against asthma, improvement is still needed to achieve increased potency of the powder formulation and a reduced number of capsules containing powder to be inhaled. In this study, high-potency amorphous powder formulations containing GDC-0214 nanoaggregates for dry powder inhalation were developed using particle engineering technology, thin film freezing (TFF). A high dose per capsule was successfully achieved by enhancing the solubility of GDC-0214 and powder conditioning. Lactose and/or leucine as excipients exhibited optimum stability and aerosolization of GDC-0214 nanoaggregates, and aerosolization of the dose was independent of air flow through the device between 2 and 6 kPa pressure drops. In the rat PK study, formulation F20, which contains 80% GDC-0214 and 20% lactose, resulted in the highest AUC0-24h in the lungs with the lowest AUC0-24h in the plasma that corresponds to a 4.8-fold higher ratio of the lung-to-plasma exposures compared to micronized crystalline GDC-0214 powder administered by dry powder inhalation. Therefore, GDC-0214 nanoaggregates produced by TFF provided an improved dry powder for inhalation that can lead to enhanced therapeutic efficacy with a lower risk of systemic toxicity.

Glycan-Driven Formation of Raft-Like Domains with Hierarchical Periodic Nanoarrays on Dendrimersome Synthetic Cells.

The accurate spatial segregation into distinct phases within cell membranes coordinates vital biochemical processes and functionalities in living organisms. One of nature's strategies to localize reactivity is the formation of dynamic raft domains. Most raft models rely on liquid-ordered L0 phases in a liquid-disordered Ld phase lacking correlation and remaining static, often necessitating external agents for phase separation. Here, we introduce a synthetic system of bicomponent glycodendrimersomes coassembled from Janus dendrimers and Janus glycodendrimers (JGDs), where lactose-lactose interactions exclusively drive lateral organization. This mechanism results in modulated phases across two length scales, yielding raft-like microdomains featuring nanoarrays at the nanoscale. By varying the density of lactose and molecular architecture of JGDs, the nanoarray type and size, shape, and spacing of the domains were controlled. Our findings offer insight into the potential primordial origins of rudimentary raft domains and highlight the crucial role of glycans within the glycocalyx.

Enzymatically Oxidized Carbohydrates As Dicarbonyl Biobased Cross-Linkers for Polyamines.

Carbonyl cross-linkers are used to modify textiles and form resins, and are produced annually in megatonne volumes. Due to their toxicity toward the environment and human health, however, less harmful biobased alternatives are needed. This study introduces carbonyl groups to lactose and galactose using galactose oxidase from Fusarium graminearum (FgrGalOx) and pyranose dehydrogenase from Agaricus bisporus (AbPDH1) to produce four cross-linkers. Differential scanning calorimetry was used to compare cross-linker reactivity, most notably resulting in a 34 °C decrease in reaction peak temperature (72 °C) for FgrGalOx-oxidized galactose compared to unmodified galactose. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and proton nuclear magnetic resonance (1H NMR) spectroscopy were used to verify imine formation and amine and aldehyde depletion. Cross-linkers were shown to form gels when mixed with polyallylamine, with FgrGalOx-oxidized lactose forming gels more effectively than all other cross-linkers, including glutaraldehyde. Further development of carbohydrate cross-linker technologies could lead to their adoption in various applications, including in adhesives, resins, and textiles.

Optimizing microbioreactor cultivation strategies for Trichoderma reesei: from batch to fed-batch operations.

BACKGROUND: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. RESULTS: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in ß -glucosidase activity compared to batch processes using the same type and amount of carbon sources. CONCLUSION: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology.

Effects of Three Kinds of Carbohydrate Pharmaceutical Excipients-Fructose, Lactose and Arabic Gum on Intestinal Absorption of Gastrodin through Glucose Transport Pathway in Rats.

BACKGROUND: Some glucoside drugs can be transported via intestinal glucose transporters (IGTs), and the presence of carbohydrate excipients in pharmaceutical formulations may influence the absorption of them. This study, using gastrodin as probe drug, aimed to explore the effects of fructose, lactose, and arabic gum on intestinal drug absorption mediated by the glucose transport pathway. METHODS: The influence of fructose, lactose, and arabic gum on gastrodin absorption was assessed via pharmacokinetic experiments and single-pass intestinal perfusion. The expression of sodium-dependent glucose transporter 1 (SGLT1) and sodium-independent glucose transporter 2 (GLUT2) was quantified via RT‒qPCR and western blotting. Alterations in rat intestinal permeability were evaluated through H&E staining, RT‒qPCR, and immunohistochemistry. RESULTS: Fructose reduced the area under the curve (AUC) and peak concentration (Cmax) of gastrodin by 42.7% and 63.71%, respectively (P < 0.05), and decreased the effective permeability coefficient (Peff) in the duodenum and jejunum by 58.1% and 49.2%, respectively (P < 0.05). SGLT1 and GLUT2 expression and intestinal permeability remained unchanged. Lactose enhanced the AUC and Cmax of gastrodin by 31.5% and 65.8%, respectively (P < 0.05), and increased the Peff in the duodenum and jejunum by 33.7% and 26.1%, respectively (P < 0.05). SGLT1 and GLUT2 levels did not significantly differ, intestinal permeability increased. Arabic gum had no notable effect on pharmacokinetic parameters, SGLT1 or GLUT2 expression, or intestinal permeability. CONCLUSION: Fructose, lactose, and arabic gum differentially affect intestinal drug absorption through the glucose transport pathway. Fructose competitively inhibited drug absorption, while lactose may enhance absorption by increasing intestinal permeability. Arabic gum had no significant influence.

Gram-scale chemical synthesis of galactosyllactoses and their impact on infant gut microbiota in vitro.

Galactooligosaccharides (GOS) are widely used as a supplement in infant nutrition to mimic the beneficial effects found in prebiotic human milk oligosaccharides (HMOs). However, the complexity of the GOS mixture makes it challenging to ascertain which of the GOS components contribute most to their health benefits. Galactosyllactoses (GLs) are lactose-based trisaccharides containing a ß-galactopyranosyl residue at the 3'-position (3'galactosyllactose, 3'-GL), 4'-position (4'-galactosyllactose, 4'-GL), or the 6'-position (6'-galactosyllactose, 6'-GL). These GLs are of particular interest as they are present in both GOS mixtures and human milk at early stages of lactation. However, research on the potential health benefits of these individual GLs has been limited. Gram quantities are needed to assess their health benefits but these GLs are not readily available at this scale. In this study, we report the gram-scale chemical synthesis of 3'-GL, 4'-GL, and 6'-GL. All three galactosyllactoses were obtained on a gram scale in good purity from cheap and commercially available lactose. Furthermore, in vitro incubation of GLs with infant faecal microbiota demonstrates that the GLs were able to increase the abundance of Bifidobacterium and stimulate short chain fatty acid production.

Synthesis and unexpected binding of monofluorinated N,N'-diacetylchitobiose and LacdiNAc to wheat germ agglutinin.

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Added Value of 13C Analysis in Breath Tests in H2-Negative Subjects to Diagnose Lactose Malabsorption: A Proof of Concept Study.

INTRODUCTION: Diagnosing lactose malabsorption is usually based on hydrogen excretion in breath after a lactose challenge. However, a proportion of subjects with lactose malabsorption will not present a rise in hydrogen. Measuring excretion of methane or stable isotope labeled 13CO2 after ingestion of 13C-lactose has been proposed to mitigate this problem. OBJECTIVE: The aim of the study was to assess the performance of measuring methane and 13CO2 in individuals with normal hydrogen excretion compared to a genetic lactase non-persistence test. METHODS: Individuals referred for lactose breath testing and healthy controls were included. Participants received 13C-enriched lactose, performed breath testing, and underwent genotyping for a marker of lactase non-persistence (13910C*T). Using genotype as gold standard, the performance of measuring methane and 13CO2 excretion was assessed. RESULTS: 151 subjects participated in the study, 50 of which presented a lactase non-persistent genotype. Of these, 72% were correctly diagnosed through hydrogen excretion of ≥ 20 ppm above baseline. In subjects with normal hydrogen excretion, cumulative 13C excretion had an area under the curve (AUC) of the receiver operating characteristics (ROC) curve of 0.852. Sensitivity was 93% and specificity was 51% for the current cutoff of 14.5%. The optimal cutoff was 12.65% (sensitivity 93%, specificity 70%). The ROC curve of peak methane had an AUC of 0.542 (sensitivity of 14%, specificity of 91% for cutoff ≥ 10 ppm). CONCLUSIONS: In individuals with genetically demonstrated lactase non-persistence and negative hydrogen breath test, the use of 13C-lactose with measurement of 13CO2 excretion and hydrogen is a well-performing test to detect the lactose malabsorption and performs better than methane in our cohort.

Correlating sugar transporter expression and activities to identify transporters for an orphan sugar substrate.

Filamentous fungi like Neurospora crassa are able to take up and metabolize important sugars present, for example, in agricultural and human food wastes. However, only a fraction of all putative sugar transporters in filamentous fungi has been characterized to date, and for many sugar substrates, the corresponding transporters are unknown. In N. crassa, only 14 out of the 42 putative major facilitator superfamily (MFS)-type sugar transporters have been characterized so far. To uncover this hidden potential for biotechnology, it is therefore necessary to find new strategies. By correlation of the uptake profile of sugars of interest after different induction conditions with the expression profiles of all 44 genes encoding predicted sugar transporters in N. crassa, together with an exhaustive phylogenetic analysis using sequences of characterized fungal sugar transporters, we aimed to identify transporter candidates for the tested sugars. Following this approach, we found a high correlation of uptake rates and expression strengths for many sugars with dedicated transporters, like galacturonic acid and arabinose, while the correlation is loose for sugars that are transported by several transporters due to functional redundancy. Nevertheless, this combinatorial approach allowed us to elucidate the uptake system for the disaccharide lactose, a by-product of the dairy industry, which consists of the two main cellodextrin transporters CDT-1 and CDT-2 with a minor contribution of the related transporter NCU00809. Moreover, a non-MFS transporter involved in glycerol transport was also identified. Deorphanization of sugar transporters or identification of transporters for orphan sugar substrates by correlation of uptake kinetics with transporter expression and phylogenetic information can thus provide a way to optimize the reuse of food industry by-products and agricultural wastes by filamentous fungi in order to create economic value and reduce their environmental impact. KEY POINTS: • The Neurospora crassa genome contains 30 uncharacterized putative sugar transporter genes. • Correlation of transporter expression and sugar uptake profiles can help to identify transporters for orphan sugar substrates. • CDT-1, CDT-2, and NCU00809 are key players in the transport of the dairy by-product lactose in N. crassa.

Kinetics and products of Thermotoga maritima ß-glucosidase with lactose and cellobiose.

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by ß-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many ß-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable ß-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the ß-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly ß(1 → 3) and ß(1 → 6) elongating activity, but also some ß(1 → 4) elongation was observed. The ß-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. KEY POINTS: • ß-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. • Thermotoga maritima ß-glucosidase has high activity and high thermostability. • Thermotoga maritima ß-glucosidase GOS contains mainly (ß1-3) and (ß1-6) linkages.

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.

Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome.

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.

Response surface methodology use in construction of polianiline-coated carbon paste electrode-based biosensor: Modification and characterization.

In this study, the effect of amperometric glucose biosensor construction and using conditions on the current response was investigated in detail applying experimental design. Polyaniline (PANI) was synthesized on the carbon paste electrode (CPE) surface using the cyclic voltammetry technique in sodium oxalate (NaOx ) electrolyte medium, and an amperometric biosensor was constructed by immobilizing glucose oxidase (GOD). Biosensor preparation (aniline, GOD and NaOx concentrations, and scan rate) and operating conditions (pH and applied potential) were optimized by Box-Behnken and optimal designs, respectively, via State Ease Design Expert 7.0.1.1 software. ANOVA analyses showed that among the biosensor preparation parameters, the NaOx concentration has the highest effect on the current measured in the presence of glucose, whereas in the optimization of pH and potential parameters applied in current measurement studies, it has been revealed that pH has a very high effect on the measured current. Several compounds, such as MWCNT, two different ionic liquids and two different organic molecules were added to carbon paste, and, among them, 2-cyanoethylpyrrole (CPy) enhanced the efficacy highly, most probably due to its polymerization in the paste and increasing the electron transfer rate of the CPE. Sucrose- and lactose-sensitive biosensors were also constructed by co-immobilizing GOD with invertase (INV) or ß-galactosidase, respectively, onto modified CPE, and sensitivities to their substrates were shown by cyclic voltammetry and impedance analysis. CPy modification caused an increase in the current values, and also Imax /KM values increased approximately 11.8, 7.83, and 2.56 times for glucose-, sucrose-, and lactose-sensitive CPEs, respectively.

Mammary fat globules as a source of mRNA to model alterations in the expression of some milk component genes during lactation in bovines.

BACKGROUND: The milk's nutritional value is determined by its constituents, including fat, protein, carbohydrates, and minerals. The mammary gland's ability to produce milk is controlled by a complex network of genes. Thereby, the fat, protein, and lactose synthesis must be boost in milk to increase milk production efficiency. This can be accomplished by fusing genetic advancements with proper management practices. Therefore, this study aimed to investigate the association between the Lipoprotein lipase (LPL), kappa casein CSN3, and Glucose transporter 1 (GLUT1) genes expression levels and such milk components as fat, protein, and lactose in different dairy breeds during different stages of lactation. METHODS: To achieve such a purpose, 94 milk samples were collected (72 samples from 36 multiparous black-white and red-white Holstein-Friesian (HF) cows and 22 milk samples from 11 Egyptian buffaloes) during the early and peak lactation stages. The milk samples were utilized for milk analysis and genes expressions analyses using non- invasive approach in obtaining milk fat globules (MFGs) as a source of Ribonucleic acid (RNA). RESULTS: LPL and CSN3 genes expressions levels were found to be significantly higher in Egyptian buffalo than Holstein-Friesian (HF) cows as well as fat and protein percentages. On the other hand, GLUT1 gene expression level was shown to be significantly higher during peak lactation than early lactation. Moreover, lactose % showed a significant difference in peak lactation phase compared to early lactation phase. Also, fat and protein percentages were significantly higher in early lactation period than peak lactation period but lactose% showed the opposite pattern of Egyptian buffalo. CONCLUSION: Total RNA can be successfully obtained from MFGs. The results suggest that these genes play a role in glucose absorption and lactose synthesis in bovine mammary epithelial cells during lactation. Also, these results provide light on the differential expression of these genes among distinct Holstein-Friesian cow breeds and Egyptian buffalo subspecies throughout various lactation phases.