Glucose Units Research Articles

Driving an in vitro multienzymatic cascade of laminaribiose biosynthesis from non-food cellulose with balancing the precursor supply

High-valued disaccharide laminaribiose, derived from two glucose units, is widely used in the pharmaceutical, cosmetic and food industries. The technology for laminaribiose synthesis from non-food feedstock cellulose instead of sucrose, glucose and starch was developed. First, we investigated a two-enzyme cascade converting cellobiose to laminaribiose by cellobiose phosphorylase and laminaribiose phosphorylase and 0.47 mol/mol cellobiose yield was obtained with cellobiose. An in vitro enzymatic cascade composed of cellobiohydrolase, cellodextrin phosphorylase, cellobiose phosphorylase and laminaribiose phosphorylase was designed to synthesize laminaribiose from cellulose. Laminaribiose production reached 2.35 and 1.23 g/L in pathways I & II, respectively. Furthermore, another multienzyme cascade was designed to eliminate the imbalance of glucose and glucose-1-p supply and laminaribiose yield finally reached 72% of that yield from cellobiose. This in vitro enzymatic cascades for laminaribiose biosynthesis not only widen the utilization of the non-food cellulose, but also provide a model that improves the economy of lignocellulosic biorefineries.

Changes of IgG N-Glycosylation in Thyroid Autoimmunity: The Modulatory Effect of Methimazole in Graves' Disease and the Association With the Severity of Inflammation in Hashimoto's Thyroiditis.

The N-glycome of immunoglobulin G (IgG), the most abundant glycoprotein in human blood serum, reflects pathological conditions of autoimmunity and is sensitive to medicines applied in disease therapy. Due to the high sensitivity of N-glycosylation, the IgG N-glycan profile may serve as an indicator of an ongoing inflammatory process. The IgG structure and its effector functions are strongly dependent on the composition of N-glycans attached to the Fc fragment, and the binding of antigens is regulated by Fab sugar moieties. Because of the crucial role of N-glycans in IgG function, remodeling of its N-oligosaccharides can induce pathological changes that ultimately contribute to the development of autoimmunity; restoration of their physiological structure is critical to the reduction of disease symptoms. Our recently published data have shown that the pathology of autoimmune thyroid diseases (AITDs), including Hashimoto’s thyroiditis (HT) and Graves’ disease (GD), is accompanied by alterations of the composition of IgG N-glycans. The present study is a more in-depth investigation of IgG glycosylation in both AITDs, designed to determine the relationship between the severity of thyroid inflammation and IgG N-glycan structures in HT, and to assess the impact of immunosuppressive therapy on the N-glycan profile in GD patients. The study material consisted of human serum samples collected from donors with elevated anti-thyroglobulin (Tg) and/or anti-thyroperoxidase (TPO) IgGs without symptoms of hypothyroidism (n=68), HT patients characterized by high autoantibody titers and advanced destruction of the thyroid gland (n=113), GD patients with up-regulated IgG against thyroid-stimulating hormone receptor (TSHR) before (n=62) and after (n=47) stabilization of TSH level as a result of methimazole therapy (study groups), and healthy donors (control group, n=90). IgG was isolated from blood serum using protein G affinity chromatography. N-glycans were released from IgG by PNGase F digestion and analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) after 2-aminobenzamide (2-AB) labeling. UPLC-MS chromatograms were integrated into 25 peaks (GP) in the Waters UNIFI Scientific Information System, and N-glycans were assigned based on the glucose unit values and mass-to-charge ratios (m/z) of the detected ions. The Kruskal-Wallis non-parametric test was used to determine the statistical significance of the results (p<0.05). The obtained results suggest that modifications of IgG sialylation, galactosylation and core-fucosylation are associated with the severity of HT symptoms. Methimazole therapy implemented in GD patients affected the IgG N-glycan profile; as a result, the content of the sialylated and galactosylated oligosaccharides with core fucose differed after treatment. Our results suggest that N-glycosylation of IgG undergoes dynamic changes during the intensification of thyroiditis in HT, and that in GD autoimmunity it is affected significantly by immunosuppressive therapy.

Exopolysaccharide Produced by Probiotic Bacillus albus DM-15 Isolated From Ayurvedic Fermented Dasamoolarishta: Characterization, Antioxidant, and Anticancer Activities.

An exopolysaccharide (EPS) was purified from the probiotic bacterium Bacillus albus DM-15, isolated from the Indian Ayurvedic traditional medicine Dasamoolarishta. Gas chromatography-mass spectrophotometry and nuclear magnetic resonance (NMR) analyses revealed the heteropolymeric nature of the purified EPS with monosaccharide units of glucose, galactose, xylose, and rhamnose. Size-exclusion chromatography had shown the molecular weight of the purified EPS as around 240 kDa. X-ray powder diffraction analysis confirmed the non-crystalline amorphous nature of the EPS. Furthermore, the purified EPS showed the maximum flocculation activity (72.80%) with kaolin clay and emulsification activity (67.04%) with xylene. In addition, the EPS exhibits significant antioxidant activities on DPPH (58.17 ± 0.054%), ABTS (70.47 ± 0.854%) and nitric oxide (58.92 ± 0.744%) radicals in a concentration-dependent way. Moreover, the EPS showed promising cytotoxic activity (20 ± 0.97 μg mL–1) against the lung carcinoma cells (A549), and subsequent cellular staining revealed apoptotic necrotic characters in damaged A549 cells. The EPS purified from the probiotic strain B. albus DM-15 can be further studied and exploited as a potential carbohydrate polymer in food, cosmetic, pharmaceutical, and biomedical applications.

Purification, biochemical characterization, and molecular cloning of cellulase from Bacillus licheniformis strain Z9 isolated from soil

BackgroundCellulose is the most prevalent biomass and renewable energy source in nature. The hydrolysis of cellulosic biomass to glucose units is essential for the economic exploitation of this natural resource. Cellulase enzyme, which is largely generated by bacteria and fungus, is commonly used to degrade cellulose. Cellulases are used in a variety of industries, including bioethanol manufacturing, textiles, detergents, drugs, food, and paper. As part of our quest to find an efficient biocatalyst for the hydrolysis of cellulosic biomass, we describe the amplification, cloning, and sequencing of cellulase (cel9z) from Bacillus licheniformis strain Z9, as well as the characterization of the resulting enzyme. ResultsCellulase was partially purified from B. licheniformis strain Z9 using (NH4)2SO4 precipitation and Sephadex G-100 gel column chromatography with 356.5 U/mg specific activity, 2.1-purification fold, and 3.07 % yield. The nucleotide sequence of the cellulase gene was deposited to the GenBank, B. licheniformis strain Z9 cellulase (cel9z) gene, under accession number MK814929. This corresponds to 1453 nucleotides gene and encodes for a protein composed of 484 amino acids. Comparison of deduced amino acids sequence to other related cellulases showed that the enzyme cel9z can be classified as a glycoside hydrolase family 9. SDS-PAGE analysis of the purified enzyme revealed that the molecular mass was 54.5 kDa. The optimal enzyme activity was observed at pH 7.4 and 30 °C. The enzyme was found to be strongly inhibited by Mg2+ and Na+, whereas strongly activated by Fe3+, Cu2+, and Ca2+. ConclusionsB. licheniformis strain Z9 and its cellulase gene can be further utilized for recombinant production of cellulases for industrial application.

Cryoprotective Metabolites Are Sourced from Both External Diet and Internal Macromolecular Reserves during Metabolic Reprogramming for Freeze Tolerance in Drosophilid Fly, Chymomyza costata.

Many cold-acclimated insects accumulate high concentrations of low molecular weight cryoprotectants (CPs) in order to tolerate low subzero temperatures or internal freezing. The sources from which carbon skeletons for CP biosynthesis are driven, and the metabolic reprogramming linked to cold acclimation, are not sufficiently understood. Here we aim to resolve the metabolism of putative CPs by mapping relative changes in concentration of 56 metabolites and expression of 95 relevant genes as larvae of the drosophilid fly, Chymomyza costata transition from a freeze sensitive to a freeze tolerant phenotype during gradual cold acclimation. We found that C. costata larvae may directly assimilate amino acids proline and glutamate from diet to acquire at least half of their large proline stocks (up to 55 µg per average 2 mg larva). Metabolic conversion of internal glutamine reserves that build up in early diapause may explain the second half of proline accumulation, while the metabolic conversion of ornithine and the degradation of larval collagens and other proteins might be two additional minor sources. Next, we confirm that glycogen reserves represent the major source of glucose units for trehalose synthesis and accumulation (up to 27 µg per larva), while the diet may serve as an additional source. Finally, we suggest that interconversions of phospholipids may release accumulated glycero-phosphocholine (GPC) and -ethanolamine (GPE). Choline is a source of accumulated methylamines: glycine-betaine and sarcosine. The sum of methylamines together with GPE and GPC represents approximately 2 µg per larva. In conclusion, we found that food ingestion may be an important source of carbon skeletons for direct assimilation of, and/or metabolic conversions to, CPs in a diapausing and cold-acclimated insect. So far, the cold-acclimation- linked accumulation of CPs in insects was considered to be sourced mainly from internal macromolecular reserves.

Two UDP-Glycosyltransferases Catalyze the Biosynthesis of Bitter Flavonoid 7-O-Neohesperidoside through Sequential Glycosylation in Tea Plants.

Flavonoid glycosides are typical bitter and astringent tasting compounds that contribute to the taste of tea beverages. However, the genes that contribute to the biosynthesis of bitter compounds (e.g., flavanone 7-O-neohesperidoside) in tea plants have yet to be identified. In this study, we identified 194 UDP-glycosyltransferases (UGTs) from the tea transcriptome database. Among them, two genes, CsUGT75L12 and CsUGT79B28, encoding flavonoid 7-O-glycosyltransferase and 7-O-glucoside(1→2)rhamnosyltransferase, respectively, were identified from Camellia sinensis. In vitro, the purified recombinant enzyme rCsUGT75L12 specifically transports the glucose unit from UDP-glucose to the 7-OH position of the flavonoid to produce the respective 7-O-glucoside. rCsUGT79B28 regiospecifically transfers a rhamnose unit from UDP-rhamnose to the 2″-OH position of flavonoid 7-O-glucosides to produce flavonoid 7-O-di-glycosides. Additionally, the expression profiles of the two CsUGTs were correlated with the accumulation patterns of 7-O-glucoside and 7-O-neohesperidoside, respectively, in tea plants. These results indicated that the two CsUGTs are involved in the biosynthesis of bitter flavonoid 7-O-neohesperidoside through the sequential glucosylation and rhamnosylation of flavonoids in C. sinensis. Taken together, our findings provided not only molecular insights into flavonoid di-glycoside metabolism in tea plants but also crucial molecular markers for controlling the bitterness and astringent taste of tea.

Safety evaluation of glucosylated steviol glycosides as a food additive in different food categories.

The EFSA Panel on Food Additive and Flavourings (FAF) assessed the safety of glucosylated steviol glycosides proposed for use as a new food additive in different food categories. Glucosylated steviol glycosides consist of a mixture of glucosylated steviol glycosides, containing 1–20 additional glucose units bound to the parent steviol glycosides. Glucosylated steviol glycosides consist of not less than 95% (on dry, dextrin‐free, basis) of total steviol glycosides, comprised of glucosylated and parent steviol glycosides. Glucosylated steviol glycosides are produced via enzymatic bioconversion using cyclomaltodextrin glucanotransferase (CGTase) (EC 2.4.1.19), derived from a non‐genetically modified strain of Anoxybacillus caldiproteolyticus, that catalyses the transfer of glucose from starch to steviol glycosides mixtures isolated from the dried leaves of Stevia Rebaudiana. The Panel considered that the metabolism of glucosylated steviol glycosides is sufficiently similar to the already authorised steviol glycosides, and thus, the toxicological data previously assessed by the ANS Panel for steviol glycosides (E 960) were considered to support their safety as food additive. The existing acceptable daily intake (ADI) for steviol glycosides (E 960) of 4 mg/kg body weight (bw) per day expressed as steviol can also be applied to glucosylated steviol glycosides. The Panel concluded that there is no safety concern for the use of glucosylated steviol glycosides as a new food additive at the proposed use and use levels. The Panel recommended some modifications to the specifications proposed by the applicant for glucosylated steviol glycosides with respect to the assay, the definition of the proposed new food additive and the proposed maximum limits for arsenic.

Characterization of Monoclonal Antibody Glycan Heterogeneity Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

Glycosylation is a critical quality attribute of monoclonal antibody (mAb) therapeutics. Hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) is an invaluable technology for the characterization of protein glycosylation. HILIC/MS-based glycan analysis relies on the library search using Glucose Units (GU) and accurate mass (AM) as the primary search parameters for identification. However, GU-based identifications are gradient-dependent and are not suitable for applications where separation gradients need to be optimized to analyze complex samples or achieve higher throughput. Additionally, the workflow requires calibration curves (using dextran ladder) to be generated for each analysis campaign, which in turn, are used to derive the GU values of the separated glycan species. To overcome this limitation, we employed a two-step strategy for targeted glycan analysis of a mAb expressed in Chinese Hamster Ovary (CHO) cells. The first step is to create a custom library of the glycans of interest independent of GU values (thereby eliminating the need for a calibration curve) and instead uses AM and retention time (RT) as the primary search variables. The second step is to perform targeted glycan screening using the custom-built library. The developed workflow was applied for targeted glycan analysis of a mAb expressed in CHO for 1) cell line selection 2) characterizing the day-wise glycan evolution in a model mAb during a fed-batch culture, 3) assessing the impact of different media conditions on glycosylation, and 4) evaluating the impact of two different process conditions on glycosylation changes in a model mAb grown in a bioreactor. Taken together, the data presented in this study provides insights into the sources of glycan heterogeneity in a model mAb that are seen during its commercial manufacturing.

Structural and bioactive characteristics of a dextran produced by Lactobacillus kunkeei AK1

In this study, structural and techno-functional characteristics of an exopolysaccharide (EPS) produced by Lactobacillus kunkeei AK1 were determined. High-performance liquid chromatography (HPLC) analysis demonstrated that EPS AK1 was composed of only glucose units. 1H and 13C Nuclear magnetic resonance (NMR) analysis revealed that EPS AK1 was a dextran type EPS containing 4.78% (1 → 4)-linked α-d-glucose branches. The molecular weight of EPS AK1 was determined to be 45 kDa by Gel Permeation Chromatography (GPC) analysis. A high level of thermal stability up to 280 °C was determined for dextran AK1 detected by Differential scanning calorimetry (DSC) and Thermogravimetric analysis (TGA). Dextran AK1 appeared as regular spheres with compact morphology and as irregular particles in the solution with no clear cross-linking between the chains of the polysaccharide observed by Scanning electron microscopy (SEM) and Atomic force microscopy (AFM) analysis, respectively. X-ray diffraction analysis (XRD) analysis demonstrated that dextran AK1 had a crystalline structure. A relatively strong antioxidant activity was observed for dextran AK1 determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging and cupric reducing antioxidant capacity (CUPRAC) tests. Finally, only a digestion ratio of 3.1% was observed for dextran AK1 following the in vitro simulated gastric digestion test.

Pilot-scale production of exopolysaccharide from Leuconostoc pseudomesenteroides XG5 and its application in set yogurt

Exopolysaccharide from Leuconostoc pseudomesenteroides XG5 (XG5 EPS) is a linear dextran that is built by glucose units via α-1,6 glycosidic bond. The primary objective of this study was to investigate the yield of XG5 EPS and its application in set yogurt. In laboratory scale, the culture conditions of XG5 EPS production were optimized using the L9 (33) orthogonal test. Here, the optimized yield of XG5 EPS was 26.02 g/L under the conditions of 100 g/L sucrose, initial pH 7.0, 25°C incubation, and 100 rpm for 36 h in a shaking flask. Based on the optimized parameters of laboratory scale, a pilot fed-batch fermentation was performed in a 50-L bioreactor with an adjusted agitation speed of 20 rpm. The XG5 EPS yield reached 40.07 g/L in fed-batch fermentation, which was 54% higher than that achieved in laboratory scale. In addition, XG5 EPS was added into set yogurt to investigate its effect on the stability of set yogurt. Our data demonstrated that the XG5 EPS improved the water-holding capacity, texture profile, and viscosity of set yogurt during cold storage compared with the controls. In particular, addition of 0.5% XG5 EPS increased the structure of 3-dimensional network of set yogurt, which eventually improved the physical stability of the set yogurt. Overall, this study provided new insights for exploring the pilot scale production and application of dextran.

Preparation of high solid content oxidized starch by acid pretreatment–H2O2 oxidation and its performance as the ligand in zirconium tanning

Highly-oxidized starch (HOS) is an excellent ligand for zirconium tanning in leather making. However, oxidation reaction can only be undertaken in diluted solution due to the high viscosity of starch, which leads to high oxidant consumption and low solid content of HOS, and therefore greatly limits its industrial application. Starch was predegraded by using HCl followed by H2O2 oxidation for preparing a practical oxidized starch (OST) ligand with high solid content. HCl (5%) remarkably reduced the viscosity of starch and simplified the subsequent oxidation, resulting in a decrease in H2O2 dosage from 60% to 40% and an increase in solid content from 10% to 38%. HCl broke the α-(1–4) glycosidic bond between glucose units, and H2O2 oxidation led to the generation of abundant carboxyl groups in OST. The OST with moderate molecular weight (Mw 5609 g/mol) and carboxyl content (6.48 mmol/g) made the tanning performance of OST–Zr comparable to those of HOS–Zr and chrome tanning agents. Therefore, this work gives new insights into the crop-polysaccharides based ligands application and exhibits huge potential for promoting industrial applications of zirconium tanning.

Construction of an InstantPC-derivatized glycan glucose unit database: A foundation work for high-throughput and high-sensitivity glycomic analysis.

The glycosylation profile of biotherapeutic glycoproteins is a critical quality attribute that is routinely monitored to ensure desired product quality, safety and efficacy. Additionally, as one of the most prominent and complex post-translational modifications, glycosylation plays a key role in disease manifestation. Changes in glycosylation may serve as a specific and sensitive biomarker for disease diagnostics and prognostics. However, the conventional 2-aminobenzamide-based N-glycosylation analysis procedure is time-consuming and insensitive with poor reproducibility. We have evaluated an innovative streamlined 96-well-plate-based platform utilizing InstantPC label for high-throughput, high-sensitivity glycan profiling, which is user-friendly, robust and ready for automation. However, the limited availability of InstantPC-labeled glycan standards has significantly hampered the applicability and transferability of this platform for expedited glycan structural profiling. To address this challenge, we have constructed a detailed InstantPC-labeled glycan glucose unit (GU) database through analysis of human serum and a variety of other glycoproteins from various sources. Following preliminary hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection separation and analysis, glycoproteins with complex glycan profiles were subjected to further fractionation by weak anion exchange HILIC and exoglycosidase sequential digestion for cross-validation of the glycan assignment. Hydrophilic interaction ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry was subsequently utilized for glycan fragmentation and accurate glycan mass confirmation. The constructed InstantPC glycan GU database is accurate and robust. It is believed that this database will enhance the application of the developed platform for high-throughput, high-sensitivity glycan profiling and that it will eventually advance glycan-based biopharmaceutical production and disease biomarker discovery.

Modulation of the Gal-9/TIM-3 Immune Checkpoint with α-Lactose. Does Anomery of Lactose Matter?

Simple SummaryThe disaccharide lactose is a common excipient in pharmaceutical products. In addition, the two anomers α- and β-lactose can exert immuno-modulatory effects. α-Lactose functions as a major regulator of the T-cell immunoglobulin mucin-3 (Tim-3)/Galectin-9 (Gal-9) immune checkpoint, through direct binding to the β-galactoside-binding lectin galectin-9. The blockade of TIM-3 with monoclonal antibodies or small molecules represents a promising approach to combat onco-hematological diseases, in particular myelodysplastic syndromes, and acute myeloid leukemia. Alternatively, the activity of the checkpoint can be modulated via targeting of Gal-9 with both α- and β-lactose. In fact, lactose is a quasi-pan-galectin ligand, capable of modulating the functions of most of the 16 galectin molecules. This review discusses the capacity of lactose and Gal-9 to modulate the TIM-3/Gal-9 and PD-1/PD-L1 immune checkpoints in oncology. The immuno-regulatory roles of lactose and Gal-9 are highlighted.The disaccharide lactose is an excipient commonly used in pharmaceutical products. The two anomers, α- and β-lactose (α-L/β-L), differ by the orientation of the C-1 hydroxyl group on the glucose unit. In aqueous solution, a mutarotation process leads to an equilibrium of about 40% α-L and 60% β-L at room temperature. Beyond a pharmaceutical excipient in solid products, α-L has immuno-modulatory effects and functions as a major regulator of TIM-3/Gal-9 immune checkpoint, through direct binding to the β-galactoside-binding lectin galectin-9. The blockade of the co-inhibitory checkpoint TIM-3 expressed on T cells with anti-TIM-3 antibodies represents a promising approach to combat different onco-hematological diseases, in particular myelodysplastic syndromes and acute myeloid leukemia. In parallel, the discovery and development of anti-TIM-3 small molecule ligands is emerging, including peptides, RNA aptamers and a few specifically designed heterocyclic molecules. An alternative option consists of targeting the different ligands of TIM-3, notably Gal-9 recognized by α-lactose. Modulation of the TIM-3/Gal-9 checkpoint can be achieved with both α- and β-lactose. Moreover, lactose is a quasi-pan-galectin ligand, capable of modulating the functions of most of the 16 galectin molecules. The present review provides a complete analysis of the pharmaceutical and galectin-related biological functions of (α/β)-lactose. A focus is made on the capacity of lactose and Gal-9 to modulate both the TIM-3/Gal-9 and PD-1/PD-L1 immune checkpoints in oncology. Modulation of the TIM-3/Gal-9 checkpoint is a promising approach for the treatment of cancers and the role of lactose in this context is discussed. The review highlights the immuno-regulatory functions of lactose, and the benefit of the molecule well beyond its use as a pharmaceutical excipient.

Preparation and Characterization of Super-Absorbing Gel Formulated from κ-Carrageenan-Potato Peel Starch Blended Polymers.

κ-carrageenan is useful for its superior gelling, hydrogel, and thickening properties. The purpose of the study was to maximize the hydrogel properties and water-absorbing capacity of κ-carrageenan by blending it with starch from potato peels to be used as safe and biodegradable water-absorbent children’s toys. The prepared materials were analyzed using FTIR and Raman spectroscopy to analyze the functional groups. Results showed that there was a shift in the characteristic peaks of starch and κ-carrageenan, which indicated their proper reaction during blend formation. In addition, samples show a peak at 1220 cm−1 corresponding to the ester sulfate groups, and at 1670 cm−1 due to the carbonyl group contained in D-galactose. SEM micrographs showed the presence of rough surface topology after blending the two polymers, with the appearance of small pores. In addition, the presence of surface cracks indicates the biodegradability of the prepared membranes that would result after enzymatic treatment. These results are supported by surface roughness results that show the surface of the κ-carrageenan/starch membranes became rougher after enzymatic treatment. The hydrophilicity of the prepared membranes was evaluated from contact angle (CA) measurements and the swelling ratio. The swelling ratio of the prepared membranes increased gradually as the starch ratio increased, reaching 150%, while the water-uptake capacity increased from 48 ± 4% for plain κ-carrageenan to 150 ± 5% for 1:2 κ-carrageenan/starch blends. The amylase enzyme showed an effective ability to degrade both the plain κ-carrageenan and κ-carrageenan/starch membranes, and release glucose units for up to 236 and 563, respectively. According to these results, these blends could be effectively used in making safe and biodegradable molded toys with superior water-absorbing capabilities.

Successive Application of Physicochemical and Enzymatic Treatments of Office Paper Waste for the Production of Bioethanol with Possible Using of Carbon Dioxide as an Indicator for the Determination of the Bioethanol Concentration

The projected depletion of fossil fuels is a big challenge for the scientists and researchers to find out new alternative sources of energy. Using of environmental wastes as raw materials for energy productions is a new trend that most countries are directed to. Office paper wastes are sources of pure cellulosic wastes that could be intensively used as precursors for bioethanol production. Current study is concerning by liberating glucose units from office paper waste to be used as fermentable sugars for bioethanol production. Both of physical/chemical and enzymatic hydrolysis were applied in individual and successive processes to maximize the amount of produced sugars. The obtained results showed that both of autoclaving and acidic hydrolysis were efficient than microwaving and buffer hydrolysis. Both of 10% H2SO4 and 10% HCl were effectively able to release 5 and 4.9 mg/ml of glucose units from paper waste under autoclaving, respectively. However, the successive acidic and enzymatic hydrolysis elevated the glucose concentration to 7 mg/ml after 48 h of incubation. Both of SEM and FTIR were used to characterize the paper waste before and after hydrolysis. Both techniques proved the degradation of cellulosic fibers of the tested paper matrix in addition to the formation of new hydroxyl group that indicates the enzymatic break down of the bond that link the glucose units together. Saccharomyces cerevisiae strain showed an ability to ferment the liberated glucose units into 0.12% bioethanol. The released carbon dioxide gas was estimated using GC analysis and the obtained concentration was quite equal to the measured bioethanol concentration. These data prove the possibility of using CO2 concentration as an in-direct assay that reflects the exact concentration of formed bioethanol without the need of regular sampling of the ferment that may result in accidental microbial contamination.

Dimeric architecture of maltodextrin glucosidase (MalZ) provides insights into the substrate recognition and hydrolysis mechanism

Maltodextrin glucosidase (MalZ) is a key enzyme in the maltose utilization pathway in Escherichia coli that liberates glucose from the reducing end of the short malto-oligosaccharides. Unlike other enzymes in the GH13_21 subfamily, the hydrolytic activity of MalZ is limited to maltodextrin rather than long starch substrates, forming various transglycosylation products in α-1,3, α-1,4 or α-1,6 linkages. The mechanism for the substrate binding and hydrolysis of this enzyme is not well understood yet. Here, we present the dimeric crystal structure of MalZ, with the N-domain generating a unique substrate binding groove. The N-domain bears CBM34 architecture and forms a part of the active site in the catalytic domain of the adjacent molecule. The groove found between the N-domain and catalytic domain from the adjacent molecule, shapes active sites suitable for short malto-oligosaccharides, but hinders long stretches of oligosaccharides. The conserved residue of E44 protrudes at subsite +2, elucidating the hydrolysis pattern of the substrate by the glucose unit from the reducing end. The structural analysis provides a molecular basis for the substrate specificity and the enzymatic property, and has potential industrial application for protein engineering.

Temperature-responsive ‘cloud’ with controllable self-assembled particle size for smart window application

The thermochromic material for smart window applications should simultaneously have the following comprehensive properties, namely, high visible light transmittance, low near-infrared (NIR) light transmittance, easy fluidity during casting, comfortable low phase transition temperature for the human body, and stability above the phase transition temperature. However, these performance requirements are difficult to meet simultaneously. We report a strategy for achieving the above performances simultaneously using controllable self-assembled particles from a thermochromic graft copolymer solution. By oxidoreduction (REDOX) between persulfate and hydroxyl groups on the glucose unit of sodium carboxymethyl cellulose (CMC), the thermochromic copolymer poly (2-(dimethylamino) ethyl methacrylate)-(N-isopropylacrylamide) (pDN) were grafted to CMC to prepare a target thermochromic copolymer of C-g-pDN. Owing to the introduction of positively charged ions, the self-assembled particles of C-g-pDN had a size ranging from 200 to 20,000 nm. Among them, the particle size of 248 to 4774 nm accounted for 80.1%, which is equivalent to the wide band of NIR light. On the basis of the ingenious structure of C-g-pDN, an ultralow NIR light transmittance (0.8%) above LCST and an ultrahigh luminous transmittance (95.7%) at 20 °C were realized. Also, the smart windows containing C-g-pDN have excellent luminous modulation up to 95.7%. At the same time, the smart windows were illuminated with infrared and xenon lamps. The results showed that the smart windows have excellent scattering performance to both NIR light and sunlight and can effectively reduce the indoor temperature to the human body's comfortable temperature, and do not affect the normal indoor lighting.

Development and characterization of an exopolysaccharide‐functionalized acid whey cheese (requesón) using Lactobacillus delbrueckii ssp. bulgaricus

Lactobacillus delbrueckii ssp. bulgaricus NCFB 2772 was employed to develop functionalized acid whey cheese (requesón) added with in situ produced exopolysaccharide (EPS) and tested as a carrier of commercial probiotic Lactobacillus paracasei ssp. paracasei. This EPS consists of glucose and galactose units, linked by β 1-4/6 bonds. The optimum temperature of EPS biosynthesis at pH 4.6 was 37°C (0.274 mg/mL of whey). Four different requesón formulations were prepared and analyzed in terms of texture properties and color. The addition of EPS increased the production yields, improved hardness, elasticity, and adhesiveness, and slightly decreased the L* component of color (luminosity). This study supports the utilization of acid whey wastes to obtain functional cheese such as requesón in which the EPS component can not only improve processing yields but also increase the values of hardness, elasticity and adhesiveness and slightly decrease luminosity of the product. Practical applications In this manuscript, we show the feasibility of obtaining an acid whey cheese functionalized with exopolysaccharide (EPS) produced in situ by fermentation with Lactobacillus delbrueckii. The presence of EPS improved production yield, textural properties, and worked as a carrier for Lactobacillus paracasei (probiotic).

Modification of eggshell membrane to impart biospecific properties

AbstractEggshell membrane (ESM) represents an inexpensive, environmentally friendly and nontoxic byproduct. Besides the unique mechanical properties of the material, the ESM surface is rich in functional groups susceptible to further modifications. In the current study two modification pathways were applied to enrich the ESM surface with glucopyranosyl units. One was based on the reaction of activated surface carboxylic groups with glucosamine and the other exploited the reactivity of the surface amino groups for grafting poly(ethylene glycol) (PEG) chains bearing end glucopyranosyl and benzaldehyde moieties. The functionalization of the PEG chains with molar masses 400 and 1500 g mol−1 was evidenced from 1H NMR and 13C NMR spectral data. The modified ESMs were characterized using Fourier transform infrared spectroscopy and scanning electron microscopy. The Brunauer–Emmett–Teller specific surface area and the total pore volume of ESM decreased upon modification. Decoration of ESM with glucopyranosyl units imparted protein recognition properties that were tested via binding studies with concanavalin A as model lectin. Further, grafting of glucose units via PEG linkers reduced bacterial adhesion onto the membrane surface. © 2021 Society of Industrial Chemistry.

Biological macromolecule as an eco-friendly high temperature corrosion inhibitor for P110 steel under sweet environment in NACE brine ID196: Experimental and computational approaches

The results of this study reveal the inhibitory performance of biological macromolecules, i.e., maltodextrin (MDL), on P110 steel in NACE brine ID196 solution saturated with CO2 at 50 °C. The MDL is an environmentally friendly corrosion inhibitor. The inhibitory performance of MDL was investigated using weight loss (WL) tests, electrochemical impedance spectroscopy (EIS), and potentiodynamic polarization (PDP). The mean and highest deviation from the mean (H. D) of the aforementioned measurements were determined. The results of a computational study predict that neutral MDL molecules exist in the corrosive medium. The EIS findings reveal that charge-transfer resistance is enhanced at high MDL concentrations, demonstrating the protective effect of the MDL molecule. The PDP results suggest that MDL behaves as a cathodic inhibitor. The MDL corrosion inhibition efficiency is 92.4% at 400 mgL-1. KI (5 mM) is found to enhance the corrosion inhibition efficiency to 95.2% at 300 mgL-1 MDL. The Langmuir model best describes the MDL adsorption process. The AFM results suggest that the average roughness (Ra) decreases from 43 nm (blank) to 10.5 nm using MDL to 8.2 nm using KI + MDL. The results of a contact angle study reveal that the addition of MDL causes the contact angle to increase from 61.28° (blank) to 84.08° (MDL)/92.17° (KI + MDL). The XPS results suggest that hydroxyl (–OH) groups act as strong adsorptive sites. Density functional theory (DFT) results reveal that as the number of glucose units increase, the MDL performance increases.