Enzyme Research Articles

Recent Prospects of Carbonaceous Nanomaterials-Based Laccase Biosensor for Electrochemical Detection of Phenolic Compounds.

Phenolic compounds (PhCs) are ubiquitously distributed phytochemicals found in many plants, body fluids, food items, medicines, pesticides, dyes, etc. Many PhCs are priority pollutants that are highly toxic, teratogenic, and carcinogenic. Some of these are present in body fluids and affect metabolism, while others possess numerous bioactive properties such as retaining antioxidant and antimicrobial activity in plants and food products. Therefore, there is an urgency for developing an effective, rapid, sensitive, and reliable tool for the analysis of these PhCs to address their environmental and health concern. In this context, carbonaceous nanomaterials have emerged as a promising material for the fabrication of electrochemical biosensors as they provide remarkable characteristics such as lightweight, high surface: volume, excellent conductivity, extraordinary tensile strength, and biocompatibility. This review outlines the current status of the applications of carbonaceous nanomaterials (CNTs, graphene, etc.) based enzymatic electrochemical biosensors for the detection of PhCs. Efforts have also been made to discuss the mechanism of action of the laccase enzyme for the detection of PhCs. The limitations, advanced emerging carbon-based material, current state of artificial intelligence in PhCs detection, and future scopes have also been summarized.

Fungal pretreatment of citrus waste improves the hydrolysis and acidogenesis of the organic fraction of urban solids wastes

This work shows the increase in hydrolysis and acidogenesis of the organic fraction of urban solid waste (OFUSW) by the action of fungal hydrolytic enzymes obtained by solid-state fermentation (SSF) with Aspergillus niger C25B28 on citrus wastes (CW) without sterilization or the addition of nutrients. To quantify the hydrolysis and acidogenesis of the organic fraction of urban solid waste, three treatments were studied in triplicate: OFUSW plus 10 % fresh CW (RC treatment), and OFUSW plus 10 % citrus wastes pretreated by SSF (RCA treatment). The mixtures were packed into an anaerobic hydrolytic leaching bed reactor. The results showed that SSF decreased the limonene concentration by 97 %, removing 20 % of fiber and 46 % of fat from fresh CW. Pectinases (37 U/g DM [dry matter]), cellulases (33 U/g DM), xylanases (98 U/gDM), and proteases (0.2 U/gDM) were produced. Significant differences were observed between the different treatments: the greatest reduction in total solids (24 %), the highest removal efficiency of organic matter as chemical oxygen demand (COD), and the reduction of fats (23 % and 28 %, respectively), was achieved in RCA, compared to those obtained in the reactors with OFUSW and ReC without pretreatment. The production rate of volatile fatty acids (VFA) was 1.4 gVFA/Ld for RCA, 4.5 times higher than in the reactors without pretreatment.

Inhibition Kinetics and Theoretical Studies on Zanthoxylum chalybeum Engl. Dual Inhibitors of α-Glucosidase and α-Amylase.

Compounds from Zanthoxylum chalybeum Engl. were previously reported for inhibitory activities of amylase and glucosidase enzymatic action on starch as a preliminary study toward the establishment of a management strategy against postprandial hyperglycemia, however, the inhibitory kinetics and molecular interaction of these compounds were never established. A study was thus designed to establish the inhibitory kinetics and in silico molecular interaction of α-glucosidase and α-amylase with Z. chalybeum metabolites based on Lineweaver-Burk/Dixon plot analyses and using Molecular Operating Environment (MOE) software, respectively. Skimmianine (5), Norchelerythrine (6), 6-Acetonyldihydrochelerythrine (7), and 6-Hydroxy-N-methyldecarine (8) alkaloids showed mixed inhibition against both α-glucosidase and α-amylase with comparable Ki to the reference acarbose (p > 0.05) on amylase but significantly higher activity than acarbose on α-glucosidase. One phenolic 2,3-Epoxy-6,7-methylenedioxyconiferol (10) showed a competitive mode of inhibition both on amylase and glucosidase which were comparable (p > 0.05) to the activity of acarbose. The other compounds analyzed and displayed varied modes of inhibition between noncompetitive and uncompetitive with moderate inhibition constants included chaylbemide A (1), chalybeate B (2) and chalybemide C (3), fagaramide (4), ailanthoidol (9), and sesame (11). The important residues of the proteins α-glucosidase and α-amylase were found to have exceptional binding affinities and significant interactions through molecular docking studies. The binding affinities were observed in the range of -9.4 to -13.8 and -8.0 to -12.6 relative to the acarbose affinities at -17.6 and -20.5 kcal/mol on α-amylase and α-glucosidase residue, respectively. H-bonding, π-H, and ionic interactions were noted on variable amino acid residues on both enzymes. The study thus provides the basic information validating the application of extracts of Z. chalybeum in the management of postprandial hyperglycemia. Additionally, the molecular binding mechanism discovered in this study could be useful for optimizing and designing new molecular analogs as pharmacological agents against diabetes.

Petroleum Hydrocarbon Catabolic Pathways as Targets for Metabolic Engineering Strategies for Enhanced Bioremediation of Crude-Oil-Contaminated Environments

Anthropogenic activities and industrial effluents are the major sources of petroleum hydrocarbon contamination in different environments. Microbe-based remediation techniques are known to be effective, inexpensive, and environmentally safe. In this review, the metabolic-target-specific pathway engineering processes used for improving the bioremediation of hydrocarbon-contaminated environments have been described. The microbiomes are characterised using environmental genomics approaches that can provide a means to determine the unique structural, functional, and metabolic pathways used by the microbial community for the degradation of contaminants. The bacterial metabolism of aromatic hydrocarbons has been explained via peripheral pathways by the catabolic actions of enzymes, such as dehydrogenases, hydrolases, oxygenases, and isomerases. We proposed that by using microbiome engineering techniques, specific pathways in an environment can be detected and manipulated as targets. Using the combination of metabolic engineering with synthetic biology, systemic biology, and evolutionary engineering approaches, highly efficient microbial strains may be utilised to facilitate the target-dependent bioprocessing and degradation of petroleum hydrocarbons. Moreover, the use of CRISPR-cas and genetic engineering methods for editing metabolic genes and modifying degradation pathways leads to the selection of recombinants that have improved degradation abilities. The idea of growing metabolically engineered microbial communities, which play a crucial role in breaking down a range of pollutants, has also been explained. However, the limitations of the in-situ implementation of genetically modified organisms pose a challenge that needs to be addressed in future research.

Insight into the development of versatile dentin bonding agents to increase the durability of the bonding interface

Despite the huge improvements made in adhesive technology over the past 50 years, there are still some unresolved issues regarding the durability of the adhesive interface. A complete sealing of the interface between the resin and the dentin substrate remains difficult to achieve, and it is doubtful whether an optimal interdiffusion of the adhesive system within the demineralized collagen framework can be produced in a complete and homogeneous way. In fact, it is suggested that hydrolytic degradation, combined with the action of dentin matrix enzymes, destabilizes the tooth-adhesive bond and disrupts the unprotected collagen fibrils. While a sufficient resin–dentin adhesion is usually achieved immediately, bonding efficiency declines over time. Thus, here, a review will be carried out through a bibliographic survey of scientific articles published in the last few years to present strategies that have been proposed to improve and/or develop new adhesive systems that can help prevent degradation at the adhesive interface. It will specially focus on new clinical techniques or new materials with characteristics that contribute to increasing the durability of adhesive restorations and avoiding the recurrent replacement restorative cycle and the consequent increase in damage to the tooth.

Microbial Enzyme Biotechnology to Reach Plastic Waste Circularity: Current Status, Problems and Perspectives.

The accumulation of synthetic plastic waste in the environment has become a global concern. Microbial enzymes (purified or as whole-cell biocatalysts) represent emerging biotechnological tools for waste circularity; they can depolymerize materials into reusable building blocks, but their contribution must be considered within the context of present waste management practices. This review reports on the prospective of biotechnological tools for plastic bio-recycling within the framework of plastic waste management in Europe. Available biotechnology tools can support polyethylene terephthalate (PET) recycling. However, PET represents only ≈7% of unrecycled plastic waste. Polyurethanes, the principal unrecycled waste fraction, together with other thermosets and more recalcitrant thermoplastics (e.g., polyolefins) are the next plausible target for enzyme-based depolymerization, even if this process is currently effective only on ideal polyester-based polymers. To extend the contribution of biotechnology to plastic circularity, optimization of collection and sorting systems should be considered to feed chemoenzymatic technologies for the treatment of more recalcitrant and mixed polymers. In addition, new bio-based technologies with a lower environmental impact in comparison with the present approaches should be developed to depolymerize (available or new) plastic materials, that should be designed for the required durability and for being susceptible to the action of enzymes.

Synergistic Antimicrobial Action of Lactoferrin-Derived Peptides and Quorum Quenching Enzymes.

Combined use of various antimicrobial peptides (AMPs) with enzymes that hydrolyze the signaling molecules of the resistance mechanism of various microorganisms, quorum sensing (QS), to obtain effective antimicrobials is one of the leading approaches in solving the antimicrobial resistance problem. Our study investigates the lactoferrin-derived AMPs, lactoferricin (Lfcin), lactoferampin and Lf(1-11), as potential partners for combination with enzymes hydrolyzing lactone-containing QS molecules, the hexahistidine-containing organophosphorus hydrolase (His6-OPH) and penicillin acylase, to obtain effective antimicrobial agents with a scope of practical application. The possibility of the effective combination of selected AMPs and enzymes was first investigated in silico using molecular docking method. Based on the computationally obtained results, His6-OPH/Lfcin combination was selected as the most suitable for further research. The study of physical-chemical characteristics of His6-OPH/Lfcin combination revealed the stabilization of enzymatic activity. A notable increase in the catalytic efficiency of action of His6-OPH in combination with Lfcin in the hydrolysis of paraoxon, N-(3-oxo-dodecanoyl)-homoserine lactone and zearalenone used as substrates was established. Antimicrobial efficiency of His6-OPH/Lfcin combination was determined against various microorganisms (bacteria and yeasts) and its improvement was observed as compared to AMP without enzyme. Thus, our findings demonstrate that His6-OPH/Lfcin combination is a promising antimicrobial agent for practical application.

Interaction study with DNA/HSA, anti-topoisomerase IIα, cytotoxicity and in vitro antiproliferative evaluations and molecular docking of indole-thiosemicarbazone compounds

In this work we will discuss the antiproliferative evaluation and the possible mechanisms of action of indole-thiosemicarbazone compounds LTs with anti-inflammatory activity, previously described in the literature. In this perspective, some analyzes were carried out, such as the study of binding to human serum albumin (HSA) and to biological targets: DNA and human topoisomerase IIα (topo). Antiproliferative study was performed with DU-145, Jukart, MCF-7 and T-47D tumor lines and J774A.1, besides HepG2 macrophages and hemolytic activity. In the HSA interaction tests, the highest binding constant was 3.70 × 106 M−1, referring to LT89 and in the fluorescence, most compounds, except for LT76 and LT87, promoted fluorescent suppression with the largest Stern-Volmer constant for the LT88 3.55 × 104. In the antiproliferative assay with DU-145 and Jurkat strains, compounds LT76 (0.98 ± 0.10/1.23 ± 0.32 μM), LT77 (0.94 ± 0.05/1.18 ± 0.08 μM) and LT87 (0.94 ± 0.12/0.84 ± 0.09 μM) stood out, due to their IC50 values mentioned above. With the MCF-7 and T-47D cell lines, the lowest IC50 was presented by LT81 with values of 0.74 ± 0.12 μM and 0.68 ± 0.10 μM, respectively, followed by the compounds LT76 and LT87. As well as the positive control amsacrine, the compounds LT76, LT81 and LT87 were able to inhibit the enzymatic action of human Topoisomerase IIα.

Masters of Misdirection: Peptidoglycan Glycosidases in Bacterial Growth.

The dynamic composition of the peptidoglycan cell wall has been the subject of intense research for decades, yet how bacteria coordinate the synthesis of new peptidoglycan with the turnover and remodeling of existing peptidoglycan remains elusive. Diversity and redundancy within peptidoglycan synthases and peptidoglycan autolysins, enzymes that degrade peptidoglycan, have often made it challenging to assign physiological roles to individual enzymes and determine how those activities are regulated. For these reasons, peptidoglycan glycosidases, which cleave within the glycan strands of peptidoglycan, have proven veritable masters of misdirection over the years. Unlike many of the broadly conserved peptidoglycan synthetic complexes, diverse bacteria can employ unrelated glycosidases to achieve the same physiological outcome. Additionally, although the mechanisms of action for many individual enzymes have been characterized, apparent conserved homologs in other organisms can exhibit an entirely different biochemistry. This flexibility has been recently demonstrated in the context of three functions critical to vegetative growth: (i) release of newly synthesized peptidoglycan strands from their membrane anchors, (ii) processing of peptidoglycan turned over during cell wall expansion, and (iii) removal of peptidoglycan fragments that interfere with daughter cell separation during cell division. Finally, the regulation of glycosidase activity during these cell processes may be a cumulation of many factors, including protein-protein interactions, intrinsic substrate preferences, substrate availability, and subcellular localization. Understanding the true scope of peptidoglycan glycosidase activity will require the exploration of enzymes from diverse organisms with equally diverse growth and division strategies.

DSS-induced acute colitis causes dysregulated tryptophan metabolism in brain: an involvement of gut microbiota

Inflammatory bowel disease can cause pathological changes of certain organs, including the gut and brain. As the major degradation route of tryptophan (Trp), Kynurenine (Kyn) pathway are involved in multiple pathologies of brain. This study sought to explore the effects of Dextran sulphate sodium (DSS)-induced colitis on serum and brain Trp metabolism (especially the Kyn pathway) and its mechanisms. We induced acute colitis and sub-chronic colitis with 3% DSS and 1% DSS respectively and found more severe intestinal symptoms in acute colitis than sub-chronic colitis. Both of the colitis groups altered Trp-Kyn-Kynurenic acid (Kyna) pathway in serum by regulating the expression of rate-limiting enzyme (IDO-1, KAT2). Interestingly, only 3% DSS group activated Trp-Kyn pathway under the action of metabolic enzymes (IDO-1, TDO-2 and KAT2) in brain. Furthermore, intestinal flora 16S rRNA sequencing showed significantly changes in both DSS-induced colitis groups, including microbial diversity, indicator species, and the abundance of intestinal microflora related to Trp metabolism. The functional pathways of microbiomes involved in inflammation and Trp biosynthesis were elevated after DSS treatment. Moreover, correlation analysis showed a significant association between intestinal flora and Trp metabolism (both in serum and brain). In conclusion, our study suggests that DSS-induced acute colitis causes dysregulation of Trp-Kyn-Kyna pathways of Trp metabolism in serum and brain by affecting rate-limiting enzymes and intestinal flora.

Glycogen-Degrading Activities of Catalytic Domains of α-Amylase and α-Amylase-Pullulanase Enzymes Conserved in Gardnerella spp. from the Vaginal Microbiome.

Gardnerella spp. are associated with bacterial vaginosis in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria, including Gardnerella spp. Co-occurrence of multiple species of Gardnerella is common in the vagina, and competition for nutrients such as glycogen likely contributes to the differential abundances of Gardnerella spp. Glycogen must be digested into smaller components for uptake, a process that depends on the combined action of glycogen-degrading enzymes. In this study, the ability of culture supernatants of 15 isolates of Gardnerella spp. to produce glucose, maltose, maltotriose, and maltotetraose from glycogen was demonstrated. Carbohydrate-active enzymes (CAZymes) were identified bioinformatically in Gardnerella proteomes using dbCAN2. Identified proteins included a single-domain α-amylase (EC 3.2.1.1) (encoded by all 15 isolates) and an α-amylase-pullulanase (EC 3.2.1.41) containing amylase, carbohydrate binding modules, and pullulanase domains (14/15 isolates). To verify the sequence-based functional predictions, the amylase and pullulanase domains of the α-amylase-pullulanase and the single-domain α-amylase were each produced in Escherichia coli. The α-amylase domain from the α-amylase-pullulanase released maltose, maltotriose, and maltotetraose from glycogen, and the pullulanase domain released maltotriose from pullulan and maltose from glycogen, demonstrating that the Gardnerella α-amylase-pullulanase is capable of hydrolyzing α-1,4 and α-1,6 glycosidic bonds. Similarly, the single-domain α-amylase protein also produced maltose, maltotriose, and maltotetraose from glycogen. Our findings show that Gardnerella spp. produce extracellular amylase enzymes as "public goods" that can digest glycogen into maltose, maltotriose, and maltotetraose that can be used by the vaginal microbiota. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community, but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major food source available to vaginal bacteria. The significance of our research is characterizing the activity of enzymes conserved in Gardnerella species that contribute to the ability of these bacteria to utilize glycogen.

Evaluation of local hemoglobinopathy prevalence and promotion of accurate hemoglobin A1c testing using historical data retrieval

ObjectivesHeterozygous hemoglobin variants are known to cause method- and variant-specific interference with hemoglobin A1c (HbA1c) quantitation. Less attention has been paid to the role of other hemoglobin variants in confounding HbA1c testing. Here we evaluated the frequency with which enzymatic (ENZ) and immunoassay (IA) HbA1c quantitation methods, i.e., those unable to routinely detect the presence of hemoglobin variants, were used within our healthcare system for HbA1c analysis in patients with elevated fetal hemoglobin as well as compound heterozygous and homozygous variants. Design & MethodsThis analysis was enabled by automated review of HbA1c result history, implemented to promote detection of variants prior to HbA1c result reporting. ResultsDuring a 54-week period, 319,290 HbA1c analyses were performed. We observed 110 unique patient cases (0.03% problem identification rate) in which HbA1c testing was ordered in the presence of either a homozygous or compound heterozygous hemoglobin variant or elevated hemoglobin F beyond the tolerance of the method. Among the 110 cases identified, 55 (50%) showed a compound heterozygous or homozygous hemoglobin variant while 55 (50%) showed elevated hemoglobin F. Of those cases involving a compound heterozygous or homozygous variant, 8/55 (15%) involved patients who had one or more ENZ or IA HbA1c results reported previously within our system. Of the 55 total compound heterozygous or homozygous variants identified, 37 (67%) were hemoglobin E, 10 (18%) hemoglobin S/C, 4 (7%) hemoglobin S, 2 (4%) hemoglobin C, 1 (2%) hemoglobin Camden, and 1 (2%) unidentified variant. ConclusionsExclusive use of methods unable to routinely detect the presence of hemoglobin variants may lead to reporting of HbA1c results that are not clinically meaningful.

Two genes, ANS and UFGT2, from Vaccinium spp. are key steps for modulating anthocyanin production.

Anthocyanins are a major group of red to blue spectrum plant pigments with many consumer health benefits. Anthocyanins are derived from the flavonoid pathway and diversified by glycosylation and methylation, involving the concerted action of specific enzymes. Blueberry and bilberry (Vaccinium spp.) are regarded as 'superfruits' owing to their high content of flavonoids, especially anthocyanins. While ripening-related anthocyanin production in bilberry (V. myrtillus) and blueberry (V. corymbosum) is regulated by the transcriptional activator MYBA1, the role of specific structural genes in determining the concentration and composition of anthocyanins has not been functionally elucidated. We isolated three candidate genes, CHALCONE SYNTHASE (VmCHS1), ANTHOCYANIDIN SYNTHASE (VmANS) and UDP-GLUCOSE : FLAVONOID-3-O-GLYCOSYLTRANSFERASE (VcUFGT2), from Vaccinium, which were predominantly expressed in pigmented fruit skin tissue and showed high homology between bilberry and blueberry. Agrobacterium-mediated transient expression of Nicotiana benthamiana showed that overexpression of VcMYBA1 in combination with VmANS significantly increased anthocyanin concentration (3-fold). Overexpression of VmCHS1 showed no effect above that induced by VcMYBA1, while VcUFGT2 modulated anthocyanin composition to produce delphinidin-3-galactosylrhamnoside, not naturally produced in tobacco. In strawberry (Fragaria × ananassa), combined transient overexpression of VcUFGT2 with a FLAVONOID 3´,5´-HYDROXYLASE from kiwifruit (Actinidia melanandra) modulated the anthocyanin profile to include galactosides and arabinosides of delphinidin and cyanidin, major anthocyanins in blueberry and bilberry. These findings provide insight into the role of the final steps of biosynthesis in modulating anthocyanin production in Vaccinium and may contribute to the targeted breeding of new cultivars with improved nutritional properties.

X-ray microtomography reveals the 3D enzymatic deconstruction pathway of raw lignocellulosic biomass

In the context of the development of a circular bioeconomy, plant biomass constitutes a vast potential source of renewable molecules of interest. The industrial process essentially consists in using enzymes for its deconstruction. However, we are still lacking a precise understanding of the enzymes' action on such a complex, multiscale porous material. Here, we demonstrate that X-ray microtomography tackles this question in a unique way, making it possible to follow precisely the enzymatic degradation of a model biomass, wheat straw. We perform a thorough analysis of the imaging results over the full length scale of the sample and as a function of degradation time. In particular, we observe the progressive disappearance of specific cellulose-rich cell walls, with distinct 3D heterogeneities in the enzymatic action at different times and length scales. Those 3D pieces of information about the degradation process contrast clearly with those obtained from classical 2D imaging techniques.

Lactic Acid Bacteria Metabolism: Mini-Review

Abstract: Lactic acid bacteria (LAB) are a heterogeneous group of Gram-positive bacteria found in various ecological niches: fermented and non-fermented foods, the intestinal tract and vagina of humans and animals, and the environment. They are microorganisms used as probiotics, food supplements, and fermentates due to their characteristics of fermentation and safety status. LAB are responsible for the sensory and shelf life of fermented foods, gut microbiota equilibrium, and health modulation through different metabolic activities by the action of various enzymes. These enzymes degrade the major compounds: carbohydrates, proteins, and lipids, leading to the production of new metabolites that are important to study with the aim of understanding all metabolic pathways and diverse possible applications. The present work summarizes the metabolism of LAB, including different pathways, key enzymes, and specific aspects of certain species.

A molecular movie of nucleotide addition by HIV-1 reverse transcriptase.

Historically, interactions between substrates and/or inhibitors and enzymes have been viewed in terms of binding of small molecules to relatively rigid protein targets. However, computational and experimental studies have revealed that many proteins, in particular DNA polymerases, undergo molecular motions over a wide range of timescales. Such conformational flexibility is critical for enzymatic activity, drug action and drug resistance. Moreover, contemporary structural biology approaches, such as X-ray crystallography, only have the ability to resolve the structures of thermodynamically stable species, and cannot inform on kinetic intermediates. To address this issue, we have developed a novel methodology to achieve time-resolved cryo-electron microscopy (TR cryo-EM) that, for the first time, facilitates visualization of short-lived protein conformations, including those transiently occurring during catalysis.Using TR cryo-EM, we have for the first time elucidated four intermediate kinetic states during HIV-1 reverse transcriptase (RT) mediated nucleotide (dATP) incorporation, supporting the feasibility and power of these approaches. Interestingly, we observe the presence of three Mg+2 in the active site during an intermediate state of the reaction, contrasting the kinetically stable structures of RT obtained via X-ray crystallography, where only two Mg+2 can be detected. The information derived from these studies improves our understanding of RT function, which in turn may facilitate development of new drugs.

After virus exposure, early bystander naïve CD8 T cell activation relies on NAD+ salvage metabolism.

CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.

Biochemical characterization and insights into the potency of the acidic Aspergillus niger NRC114 purified α-galactosidase in removing raffinose family oligosaccharides from soymilk yogurt

BackgroundBecause humans lack α-galactosidase, foods containing certain oligosaccharides from the raffinose family, such as soybeans and other legumes, may disrupt digestion and cause flatulence.ResultsAspergillus niger NRC114 α-galactosidase was purified using protein precipitation, gel filtration, and ion exchange chromatography steps, which resulted in a 123-fold purification. The purified enzyme was found to be 64 kDa using the SDS-PAGE approach. The optimum pH and temperature of the purified α-galactosidase were detected at pH 3.5 and 60 ºC, respectively. The pure enzyme exhibited potent acidic pH stability at pH 3.0 and pH 4.0 for 2 h, and it retained its full activity at 50 ºC and 60 ºC for 120 min and 90 min, respectively. The enzyme was activated using 2.5 mM of K+, Mg2+, Co2+, or Zn2+ by 14%, 23%, 28%, and 11%, respectively. The Km and Vmax values of the purified enzyme were calculated to be 0.401 µM and 14.65 μmol min−1, respectively. The soymilk yogurt showed an increase in its total phenolic content and total flavonoids after enzyme treatment, as well as several volatile compounds that were detected and identified using GC–MS analysis. HPLC analysis clarified the enzymatic action in the hydrolysis of raffinose family oligosaccharides.ConclusionThe findings of this study indicate the importance of A. niger NRC114 α-galactosidase enzyme for future studies, especially its applications in a variety of biological fields.

Multishell Colloidosome Platform with Sequential Gastrointestinal Resistance for On-Demand Probiotic Delivery.

Probiotic-based oral therapy can potentially prevent and treat diseases by regulating the balance of intestinal flora. However, significant loss of viability and bioactivity of probiotics before reaching the colon results in low delivery efficiency and therapeutic effects, which limits their clinical applications. Here, this work proposes a multishell colloidosome (MSC) platform with sequential gastrointestinal resistance for on-demand probiotic delivery based on biomimetic mineralization and microfluidic technology. Notably, the viability of the decorated probiotics increases 280-fold compared to that of free bacteria during preservation. Because of the sequential gastrointestinal resistance of MSC, encapsulated probiotics exhibit high viability (61%) under continuous exposure to extreme acidity, bile salt erosion, and enzymatic action, whereas free bacteria have a viability of 0%. Moreover, in vitro and in vivo studies reveal that MSC mainly releases probiotics in the colon and improves colonic colonization by probiotics to maintain the integrity of the intestinal barrier and regulate the balance of intestinal flora. Consequently, MSC significantly improves the therapeutic effect on colitis in mice. The MSC platform provides a promising delivery strategy to enhance the efficacy of orally administered probiotics.

Bioprocessing of oilseed cakes by fungi consortia: Impact of enzymes produced on antioxidants release

Oilseed cakes (OC) present high potential as feedstock for the biobased industry. Biotechnological processes allow OC valorization by the production of diverse value-added products and simultaneously altering OC structure, improving their nutritional value, and boosting OC utilization in animal feed. This work explored the use of fungi consortium of two different species as a bioprocessing approach to improve the nutritional quality of OC, obtain enzymes and antioxidants by solid-state fermentation (SSF) of sunflower cake (SFC) and rapeseed cake (RSC). Rhyzopus oryzae and Aspergillus ibericus consortium led to the highest production of cellulase (135 U/g) and β-glucosidase (265 U/g) while maximum protease (228 U/g) was obtained with A. niger and R. oryzae consortium. Maximum xylanase production (886 U/g) was observed in SSF of RSC resulting in high hemicellulose reduction. The synergistic action of lignocellulosic enzymes resulted in extracts with increased antioxidant potential with possible application as food additives against oxidative stress.