Enzymatic production of prebiotic oligosaccharides

Background: Glycogen synthase kinase-3 (GSK-3) is involved in the phosphorylation and inactivation of glycogen synthase. GSK-3 inhibitors have been associated with a variety of diseases, including Alzheimer´s disease (AD), diabetes type II, neurologic disorders, and cancer. The inhibition of GSK-3β isoforms is likely to represent an effective strategy against AD. Objective: The present work aimed to design and synthesize coumarin derivatives to explore their potential as GSK-3β kinase inhibitors. Method: The through different synthetic methods were used to prepare coumarin derivatives. The GSK-3β activity was measured through the ADP-Glo™ Kinase Assay, which quantifies the kinasedependent enzymatic production of ADP from ATP, using a coupled-luminescence-based reaction. A docking study was performed by using the crystallographic structure of the staurosporine/GSK-3β complex [Protein Data Bank (PDB) code: 1Q3D]. Results: The eleven coumarin derivatives were obtained and evaluated as potential GSK-3β inhibitors. Additionally, in silico studies were performed. The results revealed that the compounds 5c, 5d, and 6b inhibited GSK-3β enzymatic activity by 38.97–49.62% at 1 mM. The other coumarin derivatives were tested at 1 mM, 1 µM, and 1 nM concentrations and were shown to be inhibitor candidates, with significant IC50 (1.224–6.875 µM) values, except for compound 7c (IC50 = 10.809 µM). Docking simulations showed polar interactions between compound 5b and Lys85 and Ser203, clarifying the mechanism of the most potent activity. Conclusion: The coumarin derivatives 3a and 5b, developed in this study, showed remarkable activity as GSK-3β inhibitors.

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A Novel Pseudomonas geniculata AGE Family Epimerase/Isomerase and Its Application in d-Mannose Synthesis

Foods ◽

10.3390/foods9121809 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1809

Author(s):

Zhanzhi Liu ◽

Ying Li ◽

Jing Wu ◽

Sheng Chen

Keyword(s):

3D Structure ◽

Three Dimensional ◽

Optimal Temperature ◽

Reaction Conditions ◽

Enzymatic Production ◽

Physiological Properties ◽

Potential Applications ◽

Kinetics Of ◽

Optimal Reaction ◽

Gene Age

d-mannose has exhibited excellent physiological properties in the food, pharmaceutical, and feed industries. Therefore, emerging attention has been applied to enzymatic production of d-mannose due to its advantage over chemical synthesis. The gene age of N-acetyl-d-glucosamine 2-epimerase family epimerase/isomerase (AGEase) derived from Pseudomonas geniculata was amplified, and the recombinant P. geniculata AGEase was characterized. The optimal temperature and pH of P. geniculata AGEase were 60 °C and 7.5, respectively. The Km, kcat, and kcat/Km of P. geniculata AGEase for d-mannose were 49.2 ± 8.5 mM, 476.3 ± 4.0 s−1, and 9.7 ± 0.5 s−1·mM−1, respectively. The recombinant P. geniculata AGEase was classified into the YihS enzyme subfamily in the AGE enzyme family by analyzing its substrate specificity and active center of the three-dimensional (3D) structure. Further studies on the kinetics of different substrates showed that the P. geniculata AGEase belongs to the d-mannose isomerase of the YihS enzyme. The P. geniculata AGEase catalyzed the synthesis of d-mannose with d-fructose as a substrate, and the conversion rate was as high as 39.3% with the d-mannose yield of 78.6 g·L−1 under optimal reaction conditions of 200 g·L−1d-fructose and 2.5 U·mL−1P. geniculata AGEase. This novel P. geniculata AGEase has potential applications in the industrial production of d-mannose.

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Characterization and optimization of extracellular enzymes production by Aspergillus niger strains isolated from date by-products

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00145-y ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Reda Bellaouchi ◽

Houssam Abouloifa ◽

Yahya Rokni ◽

Amina Hasnaoui ◽

Nabil Ghabbour ◽

...

Keyword(s):

Aspergillus Niger ◽

Culture Medium ◽

Extracellular Enzymes ◽

Enzyme Expression ◽

Specific Enzyme ◽

Enzymatic Production ◽

High Production Rate ◽

High Production ◽

Incubation Temperatures ◽

By Products

Abstract Background This work aims to study the optimal conditions of the fermentation culture medium used for the production of extracellular enzymes (amylase, cellulase, lipase, and protease) from previously isolated Aspergillus niger strains in date by-products. Results The five most powerful isolates selected based on the zone of degradation formed on Petri plates by the substrate were subjected to the quantitative evaluation of their enzymatic production. All five strains showed almost similar API-ZYM profiles, with minor variations observed at the level of some specific enzyme expression. The production of cellulase and amylase was depending on pH and incubation temperatures. ASP2 strain demonstrated the high production rate of amylase (at pH 5 and 30 °C) and cellulase (at pH 6 and 30 °C) for 96 h of incubation. Conclusion The A. niger showed the ability to produce several extracellular enzymes and can be used in the valorization of different agroindustrial residues.

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Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol

Microbial Cell Factories ◽

10.1186/s12934-021-01569-4 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Katharina N. Schwaiger ◽

Monika Cserjan-Puschmann ◽

Gerald Striedner ◽

Bernd Nidetzky

Keyword(s):

Specific Activity ◽

Dry Mass ◽

High Yield ◽

Sucrose Phosphorylase ◽

Bifidobacterium Adolescentis ◽

Enzymatic Production ◽

Intracellular Enzyme ◽

Whole Cell ◽

Freeze Thaw ◽

Thaw Cycle

Abstract Background Glucosylglycerol (2-O-α-d-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG production has been implemented. This process uses sucrose phosphorylase (SucP)-catalyzed glycosylation of glycerol from sucrose, applying the isolated enzyme in immobilized form. A whole cell-based enzyme formulation might constitute an advanced catalyst for GG production. Here, recombinant production in Escherichia coli BL21(DE3) was compared systematically for the SucPs from Leuconostoc mesenteroides (LmSucP) and Bifidobacterium adolescentis (BaSucP) with the purpose of whole cell catalyst development. Results Expression from pQE30 and pET21 plasmids in E. coli BL21(DE3) gave recombinant protein at 40–50% share of total intracellular protein, with the monomeric LmSucP mostly soluble (≥ 80%) and the homodimeric BaSucP more prominently insoluble (~ 40%). The cell lysate specific activity of LmSucP was 2.8-fold (pET21; 70 ± 24 U/mg; N = 5) and 1.4-fold (pQE30; 54 ± 9 U/mg, N = 5) higher than that of BaSucP. Synthesis reactions revealed LmSucP was more regio-selective for glycerol glycosylation (~ 88%; position O2 compared to O1) than BaSucP (~ 66%), thus identifying LmSucP as the enzyme of choice for GG production. Fed-batch bioreactor cultivations at controlled low specific growth rate (µ = 0.05 h−1; 28 °C) for LmSucP production (pET21) yielded ~ 40 g cell dry mass (CDM)/L with an activity of 2.0 × 104 U/g CDM, corresponding to 39 U/mg protein. The same production from the pQE30 plasmid gave a lower yield of 6.5 × 103 U/g CDM, equivalent to 13 U/mg. A single freeze–thaw cycle exposed ~ 70% of the intracellular enzyme activity for GG production (~ 65 g/L, ~ 90% yield from sucrose), without releasing it from the cells during the reaction. Conclusions Compared to BaSucP, LmSucP is preferred for regio-selective GG production. Expression from pET21 and pQE30 plasmids enables high-yield bioreactor production of the enzyme as a whole cell catalyst. The freeze–thaw treated cells represent a highly active, solid formulation of the LmSucP for practical synthesis.

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Long-Term Safety and Efficacy of Prebiotic Enriched Infant Formula—A Randomized Controlled Trial

Nutrients ◽

10.3390/nu13041276 ◽

2021 ◽

Vol 13 (4) ◽

pp. 1276

Author(s):

Franka Neumer ◽

Orenci Urraca ◽

Joaquin Alonso ◽

Jesús Palencia ◽

Vicente Varea ◽

...

Keyword(s):

Infant Formula ◽

Controlled Trial ◽

Fecal Microbiota ◽

First Year ◽

Double Blind Study ◽

Double Blind ◽

Term Infants ◽

Intention To Treat ◽

Prebiotic Oligosaccharides ◽

First Year Of Life

The present study aims to evaluate the effects of an infant formula supplemented with a mixture of prebiotic short and long chain inulin-type oligosaccharides on health outcomes, safety and tolerance, as well as on fecal microbiota composition during the first year of life. In a prospective, multicenter, randomized, double-blind study, n = 160 healthy term infants under 4 months of age were randomized to receive either an infant formula enriched with 0.8 g/dL of Orafti®Synergy1 or an unsupplemented control formula until the age of 12 months. Growth, fever (>38 °C) and infections were regularly followed up by a pediatrician. Digestive symptoms, stool consistency as well as crying and sleeping patterns were recorded during one week each study month. Fecal microbiota and immunological biomarkers were determined from a subgroup of infants after 2, 6 and 12 months of life. The intention to treat (ITT) population consisted of n = 149 infants. Both formulae were well tolerated. Mean duration of infections was significantly lower in the prebiotic fed infants (p < 0.05). The prebiotic group showed higher Bifidobacterium counts at month 6 (p = 0.006), and higher proportions of Bifidobacterium in relation to total bacteria at month 2 and 6 (p = 0.042 and p = 0.013, respectively). Stools of infants receiving the prebiotic formula were softer (p < 0.05). Orafti®Synergy1 tended to beneficially impact total daily amount of crying (p = 0.0594). Supplementation with inulin-type prebiotic oligosaccharides during the first year of life beneficially modulates the infant gut microbiota towards higher Bifidobacterium levels at the first 6 months of life, and is associated with reduced duration of infections.

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A Machine Learning Approach to Study Glycosidase Activities from Bifidobacterium

Microorganisms ◽

10.3390/microorganisms9051034 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1034

Author(s):

Carlos Sabater ◽

Lorena Ruiz ◽

Abelardo Margolles

Keyword(s):

Machine Learning ◽

Supervised Classification ◽

Machine Learning Algorithms ◽

Learning Approach ◽

Human Milk Oligosaccharides ◽

Future Studies ◽

High Fiber ◽

Machine Learning Approach ◽

Prebiotic Oligosaccharides

This study aimed to recover metagenome-assembled genomes (MAGs) from human fecal samples to characterize the glycosidase profiles of Bifidobacterium species exposed to different prebiotic oligosaccharides (galacto-oligosaccharides, fructo-oligosaccharides and human milk oligosaccharides, HMOs) as well as high-fiber diets. A total of 1806 MAGs were recovered from 487 infant and adult metagenomes. Unsupervised and supervised classification of glycosidases codified in MAGs using machine-learning algorithms allowed establishing characteristic hydrolytic profiles for B. adolescentis, B. bifidum, B. breve, B. longum and B. pseudocatenulatum, yielding classification rates above 90%. Glycosidase families GH5 44, GH32, and GH110 were characteristic of B. bifidum. The presence or absence of GH1, GH2, GH5 and GH20 was characteristic of B. adolescentis, B. breve and B. pseudocatenulatum, while families GH1 and GH30 were relevant in MAGs from B. longum. These characteristic profiles allowed discriminating bifidobacteria regardless of prebiotic exposure. Correlation analysis of glycosidase activities suggests strong associations between glycosidase families comprising HMOs-degrading enzymes, which are often found in MAGs from the same species. Mathematical models here proposed may contribute to a better understanding of the carbohydrate metabolism of some common bifidobacteria species and could be extrapolated to other microorganisms of interest in future studies.

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Extraction of sugarcane bagasse arabinoxylan, integrated with enzymatic production of xylo-oligosaccharides and separation of cellulose

Biotechnology for Biofuels ◽

10.1186/s13068-021-01993-z ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Leila Khaleghipour ◽

Javier A. Linares-Pastén ◽

Hamid Rashedi ◽

Seyed Omid Ranaei Siadat ◽

Andrius Jasilionis ◽

...

Keyword(s):

Sugarcane Bagasse ◽

Sugar Content ◽

Value Added ◽

Apparent Molecular Weight ◽

Bacillus Halodurans ◽

Glycoside Hydrolase Family ◽

High Concentration ◽

Enzymatic Production ◽

Successful Separation ◽

Ft Ir

AbstractSugarcane processing roughly generates 54 million tonnes sugarcane bagasse (SCB)/year, making SCB an important material for upgrading to value-added molecules. In this study, an integrated scheme was developed for separating xylan, lignin and cellulose, followed by production of xylo-oligosaccharides (XOS) from SCB. Xylan extraction conditions were screened in: (1) single extractions in NaOH (0.25, 0.5, or 1 M), 121 °C (1 bar), 30 and 60 min; (2) 3 × repeated extraction cycles in NaOH (1 or 2 M), 121 °C (1 bar), 30 and 60 min or (3) pressurized liquid extractions (PLE), 100 bar, at low alkalinity (0–0.1 M NaOH) in the time and temperature range 10–30 min and 50–150 °C. Higher concentration of alkali (2 M NaOH) increased the xylan yield and resulted in higher apparent molecular weight of the xylan polymer (212 kDa using 1 and 2 M NaOH, vs 47 kDa using 0.5 M NaOH), but decreased the substituent sugar content. Repeated extraction at 2 M NaOH, 121 °C, 60 min solubilized both xylan (85.6% of the SCB xylan), and lignin (84.1% of the lignin), and left cellulose of high purity (95.8%) in the residuals. Solubilized xylan was separated from lignin by precipitation, and a polymer with β-1,4-linked xylose backbone substituted by arabinose and glucuronic acids was confirmed by FT-IR and monosaccharide analysis. XOS yield in subsequent hydrolysis by endo-xylanases (from glycoside hydrolase family 10 or 11) was dependent on extraction conditions, and was highest using xylan extracted by 0.5 M NaOH, (42.3%, using Xyn10A from Bacillus halodurans), with xylobiose and xylotriose as main products. The present study shows successful separation of SCB xylan, lignin, and cellulose. High concentration of alkali, resulted in xylan with lower degree of substitution (especially reduced arabinosylation), while high pressure (using PLE), released more lignin than xylan. Enzymatic hydrolysis was more efficient using xylan extracted at lower alkaline strength and less efficient using xylan obtained by PLE and 2 M NaOH, which may be a consequence of polymer aggregation, via remaining lignin interactions.

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Designing a functional rice muffin formulated with prebiotic oligosaccharides and sugar reduction

Food Bioscience ◽

10.1016/j.fbio.2020.100858 ◽

2021 ◽

Vol 40 ◽

pp. 100858

Author(s):

Cláudia Amorim ◽

Beatriz B. Cardoso ◽

Sara C. Silvério ◽

Jessica C. Silva ◽

Joana I. Alves ◽

...

Keyword(s):

Sugar Reduction ◽

Prebiotic Oligosaccharides

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Green Production of Cladribine by Using Immobilized 2′-Deoxyribosyltransferase from Lactobacillus delbrueckii Stabilized through a Double Covalent/Entrapment Technology

Biomolecules ◽

10.3390/biom11050657 ◽

2021 ◽

Vol 11 (5) ◽

pp. 657

Author(s):

Cintia Wanda Rivero ◽

Natalia Soledad García ◽

Jesús Fernández-Lucas ◽

Lorena Betancor ◽

Gustavo Pablo Romanelli ◽

...

Keyword(s):

Silica Nanoparticles ◽

Enzymatic Synthesis ◽

Calcium Alginate ◽

Covalent Immobilization ◽

Chemical Processes ◽

Lactobacillus Delbrueckii ◽

Proof Of Concept ◽

Enzymatic Production ◽

Green Production ◽

Efficient Alternative

Nowadays, enzyme-mediated processes offer an eco-friendly and efficient alternative to the traditional multistep and environmentally harmful chemical processes. Herein we report the enzymatic synthesis of cladribine by a novel 2′-deoxyribosyltransferase (NDT)-based combined biocatalyst. To this end, Lactobacillus delbrueckii NDT (LdNDT) was successfully immobilized through a two-step immobilization methodology, including a covalent immobilization onto glutaraldehyde-activated biomimetic silica nanoparticles followed by biocatalyst entrapment in calcium alginate. The resulting immobilized derivative, SiGPEI 25000-LdNDT-Alg, displayed 98% retained activity and was shown to be active and stable in a broad range of pH (5–9) and temperature (30–60 °C), but also displayed an extremely high reusability (up to 2100 reuses without negligible loss of activity) in the enzymatic production of cladribine. Finally, as a proof of concept, SiGPEI 25000-LdNDT-Alg was successfully employed in the green production of cladribine at mg scale.

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