Glycosides Research Articles

Mitigating salinity stress through interactions between microalgae and different forms (free-living & alginate gel-encapsulated) of bacteria isolated from estuarine environments

Salinity stress in estuarine environments poses a significant challenge for microalgal survival and proliferation. The interaction between microalgae and bacteria shows promise in alleviating the detrimental impacts of salinity stress on microalgae. Our study investigates this interaction by co-cultivating Chlorella sorokiniana, a freshwater microalga, with a marine growth-promoting bacterium Pseudomonas gessardii, both of which were isolated from estuary. In this study, bacteria were encapsulated using sodium alginate microspheres to establish an isolated co-culture system, preventing direct exposure between microalgae and bacteria. We evaluated microalgal responses to different salinities (5 PSU, 15 PSU) and interaction modes (free-living, gel-encapsulated), focusing on growth, photosynthesis, cellular metabolism, and extracellular polymeric substances (EPS) properties. High salinity inhibited microalgal proliferation, while gel-fixed interaction boosted Chlorella growth rate by 50.7 %. Both attached and free-living bacteria restored Chlorella's NPQ to normal levels under salt stress. Microalgae in the free-living interaction group exhibited a significantly lower respiratory rate compared to the pure algae group (−17.2 %). Increased salinity led to enhanced EPS polysaccharide secretion by microalgae, particularly in interaction groups (19.7 %). Both salt stress and interaction increased the proportion of aromatic proteins in microalgae's EPS, enhancing its stability by modulating EPS glycosidic bond C-O-C and protein vibrations. This alteration caused microalgal cells to aggregate, free-living bacteria co-culture group, and fixed co-culture group increasing by 427.5 %, 567.1 %, and 704.1 %, respectively. In gel-fixed bacteria groups, reduced neutral lipids don't accumulate starch instead, carbon redirects to cellular growth, aiding salt stress mitigation. These synergistic activities between salinity and bacterial interactions are vital in mitigating salinity stress, improving the resilience and growth of microalgae in saline conditions. Our research sheds light on the mechanisms of microalgal-bacterial interactions in coping with salt stress, offering insights into the response of estuarine microorganisms to global environmental changes and their ecological stability.

Effects of radiation dose and dose rate on alginate and the use of radiation degraded alginate for peanut

Aqueous solutions of alginate (4 %) with or without hydrogen peroxide (0–2 % H2O2) were irradiated under a gamma Co-60 source. The effect of dose rate on the radiation scission yield (Gs) of resulting irradiated alginate was determined. At the dose of 20 kGy, the G(s) value of irradiated alginate decreased with the increase dose rate, suggesting that the irradiation at a suitable dose rate could further improve the radiation chemical yield of degradation. For the alginate irradiated at the same dose rate, G(s) value increased with the increase of H2O2 concentration. Average molecular weight (Mw) and polydispersity index (PI) of irradiated alginate rapidly decreased with the increase in dose and further decreased by addition of H2O2. The oligoalginate with Mw ~ 9800 g/mol was obtained by radiation degradation of 4 % alginate solution containing 2 % H2O2 at dose of 20 kGy. Radiation scission of glycoside bonds and formation of carbonyl groups (C=O) were indicated in UV and FTIR spectra of irradiated alginate. Peanut seedlings were fertilized with alginate and oligoalginate solutions, and the results showed that all growth parameters of the treated plants were better than those of the control. Furthermore, the oligoalginate prepared by gamma irradiation can be applied as a plant growth promoter for agriculture production.

Modelling of reaction kinetics in production of hydroxy carboxylic acids by alkaline degradation of cellulosic waste

The reaction kinetics in alkaline degradation of cellulosic materials to monomeric compounds at elevated temperatures was studied. The target compounds are hydroxy carboxylic acids, preferably glucoisosaccharinic acid (GISA), that are valuable platform chemicals. This methodology, which focuses on depolymerizing cellulosic materials and transforming them into hydroxy carboxylic acids, has not been explored in existing literature. To this end, a rigorous mathematical model was developed that considers phenomena at macroscopic level (transformation of crystalline cellulose into amorphous) and at microscopic level (cleavage of glycosidic bonds). Experimental cellulose degradation data in 10 wt-% NaOH and 20–200 °C was correlated with the model. The agreement between the model results and the experimental data confirmed that the process obeys the proposed reaction pathway. Around 80 % of degradation occurs during the reactor warming up period. Analysis of rate constants indicates that GISA is not degraded into smaller hydroxy acids (SHA) at the temperatures studied. Instead, monosaccharides are converted into SHA as soon as they are produced. Conversion of crystalline cellulose into amorphous form was identified as the rate determining step.

In Silico Study on a Binding Mechanism of ssDNA Aptamers Targeting Glycosidic Bond-Containing Small Molecules.

Aptamer-based detection targeting glycoconjugates has attracted significant attention for its remarkable potential in identifying structural changes in saccharides in different stages of various diseases. However, the challenges in screening aptamers for small carbohydrates or glycoconjugates, which contain highly flexible and diverse glycosidic bonds, have hindered their application and commercialization. In this study, we investigated the binding conformations between three glycosidic bond-containing small molecules (GlySMs; glucose, N-acetylneuraminic acid, and neomycin) and their corresponding aptamers in silico, and analyzed factors contributing to their binding affinities. Based on the findings, a novel binding mechanism was proposed, highlighting the central role of the stem structure of the aptamer in binding and recognizing GlySMs and the auxiliary role of the mismatched bases in the adjacent loop. Guided by this binding mechanism, an aptamer with a higher 6'-sialyllactose binding affinity was designed, achieving a KD value of 4.54 ± 0.64 μM in vitro through a single shear and one mutation. The binding mechanism offers crucial guidance for designing high-affinity aptamers, enhancing the virtual screening efficiency for GlySMs. This streamlined workflow filters out ineffective binding sites, accelerating aptamer development and providing novel insights into glycan-nucleic acid interactions.

Emulsification performance and stabilization mechanism of okra polysaccharides with different structural properties

To systematically study the relationship between structural and emulsification properties of okra polysaccharide, OPW (okra polysaccharide extracted by water), OPH (okra polysaccharide extracted by hydrochloric acid), and OPA (okra polysaccharide extracted by alkali) were prepared, which were all Rhamnogalacturonan-I enriched polysaccharides. OPW and OPH showed the same glycosidic bonds, while the degree of methyl-esterification (DM) and protein content were significantly different. The molecular weight (Mw, 7304.39 kDa) and DM (6.54%) of OPA decreased significantly, while its (Gal + Ara)/Rha was the largest. OPW, OPH, and OPA were able to emulsify oil in water over a wide pH range (3–7) at low concentration, while OPA demonstrated the poorest emulsifying stability under thermal and storage conditions. The emulsifying properties of okra polysaccharides were regulated by multi-scale molecular structure. OPA, with the highest electrostatic repulsion and the longest side chain, formed the largest d4,3 (108.94 μm) and the thinnest interface layer (0.25 μm). OPW, with the highest acetyl content (6.33%) and protein content (14.04%), formed the thickest interfacial layer (0.97 μm) and a medium d4,3 (86.40 μm). OPH, with the highest DM (33.43%) and the largest Mw (8454.63 kDa), formed a medium interfacial thickness (0.83 μm) and the smallest d4,3 (72.30 μm), exhibiting the best emulsifying performance. The present results showed controlling factors for emulsifying properties of okra polysaccharide were hydrophobic groups and steric interactions, rather than electrostatic repulsion. Meanwhile, okra polysaccharides can be strong candidates for emulsification.

Double-Ionization damage to guanine nucleotide

Computations were carried out to investigate the mechanism of double-ionization damage for deoxyguanosine nucleotide. The results reveal that the polarization of the water environment reduces double-ionization energies and facilitates the hole transfer. Double ionization in solutions is independent of the counterion. The environment screens the contribution of the phosphate group. After double ionization, cleavage of the C-N glycosidic bond is a barrierless process in the gas phase. The barrier height of bond break is ∼ 4.0 kcal/mol in solutions. The C-N scission could be a potential damaged pathway. The counterion has a minor effect on the bond rupture.

Monosaccharide composition of lignocellulosic matrix—Optimization of microwave-assisted acid hydrolysis condition

Compositional analysis of polysaccharides is crucial in many applications of analytical chemistry, and typically involves hydrolysis as initial step, to break glycosidic bonds and release individual monosaccharide units. We developed a new method for the microwave-assisted acid hydrolysis of polysaccharides, here applied for determining the composition of the polysaccharide fraction within the lignocellulosic matrix of Pinus sylvestris. The optimisation of reaction conditions was carried out using a GC–MS method after a double-step sugar derivatization process.Under the optimized conditions, specifically, employing 2 M TFA, 40 min, and 120 °C, a high depolymerisation yield was achieved with minimal degradation. Furthermore, the amount of polysaccharides detected in pine wood aligns with the results in existing literature, substantiating the determined chemical composition.The synergy of microwave-assisted acid hydrolysis and full factorial design not only facilitated the determination of the optimal hydrolysis conditions but also enabled the assessment of the parameters influencing the polysaccharide hydrolysis process and their relationships. This approach made it feasible to achieve maximum hydrolysis efficiency while minimizing analyte degradation, employing a method that reduces environmental impact through the use of microwave energy. This strategy effectively reduced the required reaction temperature, time, and reagent quantities.

HAuCl4-mediated green synthesis of highly stable Au NPs from natural active polysaccharides: Synthetic mechanism and antioxidant property

Polysaccharide-functionalized gold nanoparticles (Polysaccharide-Au NPs) with high stability were successfully prepared by a straightforward method. Notably, the Au (III) ion acts as a strong Lewis acid to facilitate glycosidic bond breaking. Subsequently, the polysaccharide conformation was transformed to an open-chain form, exposing highly reduced aldehyde or ketone groups that reduce Au (III) to Au (0) crystal species, further growing into Au NPs. As-prepared Au NPs displayed excellent stability over a longer storage period (more than 70 days), a wide range of temperatures (25–60 °C), and pH range (3−11), varying concentrations (0–200 mM) and types of salt ions (Na+, K+, Ca2+, Mg2+), and glutathione solutions (5 mM). More interestingly, polysaccharide-Au NPs retained the antioxidant activity of polysaccharides and reduced oxidative damage at the cellular level through decreased reactive oxygen species (ROS) production. The intracellular levels of ROS pretreated with polysaccharide and polysaccharide-Au NPs were decreased 53.12–75.85 % compared to the H2O2 group, respectively. Therefore, the green synthesized Au NPs from natural active polysaccharides exhibit potential applications in biomedical fields.

KARAKTERISASI SELULASE TERMOSTABIL ASAL ISOLAT BAKTERI TANAH PERTANIAN PASANG SURUT

Cellulase is an enzyme that can break up β - (1→4) glycosidic bonds of cellulose. Generally, cellulases are classified into three major groups, i.e., endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1 .91), and β glucosidase (EC 3.2.1.21). Meanwhile, five isolates of tidal swamp soil have been isolated; thus, in this research step, we are acquainted with cellulase activity and the characterization of cellulase enzymes on various temperatures, pH, and specific substrates. The activity of an enzyme was measured using the modified Miller and Bradford methods to detect the specific characteristics of the isolate. The results showed that five isolates namely ATP-1 BKP-5, HAMML-2, PKL-2, and SPBKK-3 have activities 0.025 nKat.ml-1, 0.032 nKat.ml-1, 0.296 nKat.ml-1, 0.114 nKat.ml-1, and 0.087 nKat.ml-1, respectively. Furthermore, cellulase from isolates ATP 1, BKP-5, and PKL 2 have an optimum activity at pH 7,0; 50 oC. The isolate HAMML-2 has an optimum activity at pH 5,0; 60 oC. SPBK 3 has an optimum activity at pH 5,0; 50 oC. All of those isolates can degrade CMC, Avicell, and Whatman paper No.1 and retain the activity relative to the temperatures 50 and 60 0C as the characteristics of the thermostable enzyme.

Computational study on the inhibition mechanism of cyclodextran against GTF-SI from Streptococcus mutans focusing on the glucan-binding domain

Glucosyltransferases (GTFs) from Streptococcus mutans (S. mutans) are enzymes that synthesize glucan, a polymer that forms a biofilm causing dental caries. Cyclodextran (CI), which contains a ring structure formed from D-glucose by α-1,6 glycoside linkage, has been reported to strongly inhibit GTFs. In contrast, cyclodextrin, formed by α-1,4 glycoside linkage, has almost no inhibitory activity. In this study, we examined the inhibition mechanism of CI through computational chemistry, focusing on the glucan-binding domain of GTF-SI, which is one of GTFs produced by S. mutans. Analysis using molecular dynamics simulation and fragment molecular orbital method demonstrated a clear difference in the molecular interaction mechanism between α-1,6- and α-1,4-linked oligosaccharides. The calculated affinity of α-1,6-linked oligosaccharides was higher than that of α-1,4-linked oligosaccharides. Additionally, geometry optimization based on density functional theory revealed that three amino acids—Q1282, K1284, and S1302—play an essential role in the binding with α-1,6-linked oligosaccharides. These results successfully explain the inhibition mechanism of CI.

Stereoselective α‐Glycosylation with GlcN3 Donors Enabled Collective Syntheses of Acinetobacter baumannii Capsular Polysaccharides K43, K47 and K88 Repeating Units†

Comprehensive SummaryCollective syntheses of A. baumannii CPS K43, K47 and K88 repeating units have been accomplished via a new α‐glycosylation method with GlcN3 as donors, which features: 1) mild reaction conditions, 2) good to high yields, 3) excellent stereoselectivities. The synthetic route also highlights an orthogonal one‐pot coupling strategy on the basis of glycosyl ortho‐(1‐phenylvinyl)benzoates for stereoselective constructions of both 1,2‐trans and 1,2‐cis glycosidic bonds, precluding the issues of aglycone transfer.

A GMR enzymatic assay for quantifying nuclease and peptidase activity.

Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor's resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

Investigation of the role of sodium chloride on wheat starch multi-structure, physicochemical and digestibility properties during X-ray irradiation

In this paper, different NaCl content was added to wheat starch and then subjected to X-ray irradiation to investigate the effect of salt on starch modification by irradiation. The results showed that the degradation of wheat starch intensified with the increase in irradiation dose. When irradiated at the same dose, wheat starch with sodium chloride produced shorter chains, lower molecular weight and amylose content, and higher crystallinity, solubility, and resistant starch than wheat starch without sodium chloride. The energy generated by X-rays dissociating sodium chloride caused damage to the glycoside bonds of the starch molecule. With a further increase in the mass fraction of NaCl, the hydrogen bonds of the starch molecules were broken, and the double helix structure was depolymerized, which exacerbated the extent of irradiation-modified wheat starch. At the same time, starch molecules will be rearranged to form a more stable structure.

Hydrolysis of Cellulose using boronic-acid multifunctional carbocatalysts

Background Developingeffective and sustainable catalysts for cellulose hydrolysis is crucial for transforming this abundant biopolymer into renewable chemicals and biofuel. Carbonaceous solid acids are promising catalysts for this reaction due to their low cost, chemical and hydrothermal stability, tunable surface, and high affinity for cellulose. MethodsWe developed multifunctional carbocatalysts for cellulose hydrolysis by introducing boronic groups and weak acid sites along with high-density sulfonic acid through borylation of mesophase pitch carbon (MPC) followed by sulfonation. The functionalized MPC was characterized using various techniques, including FT-IR, XPS, SS-NMR, Raman spectroscopy, and TGA. Significant findingsThe results of surface characterization revealed the successful introduction of multifunctional groups on the peripheral surface of MPC. This carbocatalyst can effectively hydrolyze pretreated cellulose into glucose, resulting in a maximum yield of 64 % in water at 180 °C for 3 h. Boronic groups and weak acid sites could mediate the interaction of cellulose molecules with catalyst surface to enhance the catalytic hydrolysis. The apparent activation energy for breaking the glycosidic bonds using the functionalized MPC is 99 kJ/mol, which is lower than the one derived from homogeneous acids. Furthermore, the carbocatalyst is recyclable at least four cycles prior to the significant loss for reactivity.

Unraveling interplay between lignocellulosic structures caused by chemical pretreatments in enhancing enzymatic hydrolysis

To investigate the interplay between substrate structure and enzymatic hydrolysis (EH) efficiency, poplar was pretreated with acidic sodium-chlorite (ASC), 3 % sodium-hydroxide (3-SH), and 3 % sulfuric acid (3-SA), resulting in different glucose yields of 94.10 %, 74.35 %, and 24.51 %, respectively, of pretreated residues. Residues were fractionated into cellulose, lignin and unhydrolyzed residue after EH (for lignin-carbohydrate complex (LCC) analysis) and analyzed using HPLC, FTIR, XPS, CP MAS 13C NMR and 2D-NMR (Lignin and LCC analysis). After delignification, holocellulose exhibited a dramatic increase in glucose yield (74.35 % to 90.82 % for 3-SH and 24.51 % to 80.0 % for 3-SA). Structural analysis of holocellulose suggested the synergistic interplay among cellulose allomorphs to limit glucose yield. Residual lignin analysis from un/pretreated residues indicated that higher β-β' contents and S/G ratios were favorable to the inhibitory effect but unfavourable to the holocellulose digestibility and followed the trend in the following order: 3-SA (L3) > 3-SH (L2) > native-lignin (L1). Analysis of enzymatically unhydrolyzed pretreated residues revealed the presence of benzyl ether (BE1,2) LCC and phenyl glycoside (PG) bond linking to xylose (X) and mannose (M), which yielded a xylan-lignin-glucomannan network. The stability, steric hindrance and hydrophobicity of this network may play a central role in defining poplar recalcitrance.

Facile extraction of lignin-carbohydrate complexes from bamboo with formic acid aqueous solution under moderate conditions

The extraction of lignin-carbohydrate complexes (LCC) while preserving the majority of the interconnecting bonds poses a formidable challenge in characterizing the structure and exploring further applications. In this study, a straightforward approach was proposed to extract LCC from bamboo by precisely adjusting formic acid aqueous solution concentrations (70 % to 100 %) and reaction temperatures (40 to 100 °C) under normal pressure. Notably, employing a 90 % formic acid aqueous solution at 100 °C for 2 h led to an impressive LCC yield of 24.47 %. Moreover, all the LCC samples demonstrated exceptional homogeneity with a polydispersity index ≤ 1.97. Nuclear magnetic resonance analysis revealed the presence of phenyl glycosidic, benzyl ether, benzyl ester, and acetal bonds within the extracted LCC, with phenyl glycosidic bonds serving as the predominant linkages. The LCC samples displayed notable antioxidant capacity, as evidenced by their high DPPH free radical scavenging index of 1.28.

Effects of UV/H2O2 degradation on Moringa oleifera Lam. leaves polysaccharides: Composition, in vitro fermentation and prebiotic properties on gut microorganisms

Moringa oleifera Lam. leaves are a new raw food material rich in polysaccharides. These polysaccharides exhibit various biological properties, including antioxidant, hypoglycemic and immunoregulatory effects. However, the use of Moringa oleifera Lam. leaves polysaccharides (MOLP) may be limited by their large molecular weight (MW) and presence of numerous impurities, such as pigments. Research has indicated that degraded polysaccharides usually exhibit high biological activity because of changes in physical structure and chemical properties. In this study, we focused on the extraction of a degraded-modified fraction from MOLP using the Ultraviolet/ Hydrogen peroxide (UV/H2O2) method. Specifically, the physicochemical properties and glycosidic bond composition of a particular fraction (UV/H2O2 degraded Moringa oleifera Lam. leaves polysaccharides in 3 h called DMOLP-3) were investigated. In addition, in vitro simulated digestion experiments showed that DMOLP-3 was only partially degraded during gastrointestinal digestion, indicating that DMOLP-3 can be utilised by gut microorganisms. Furthermore, the prebiotic properties of MOLP and DMOLP-3 was studied using an in vitro faecal fermentation model. The results indicated that compared with MOLP, DMOLP-3 led to a decrease in both the colour and MW of the polysaccharides. In addition, this model exhibited enhanced solubility and antioxidant capabilities while also influencing the surface morphology. Moreover, DMOLP-3 can facilitate the proliferation of advantageous microorganisms and enhance the synthesis of short-chain fatty acids (SCFAs). These results provide valuable insights into the utilization of bioactive components in Moringa oleifera Lam. leaves for the intestinal health.

Evaluate the Structural and Physicochemical Properties of Exopolysaccharides Produced by Bacillus halotolerans Isolated from Locally Sourced Vegetables.

In this study, a Bacillus halotolerans (B. halotolerans) strain DT1 capable of producing exopolysaccharides (EPS) was isolated from dried cabbages of Tianjin, a local fermented vegetable product. Three distinct polysaccharide fractions were isolated from the fermentation broth of DT1, namely, DT1-0, DT1-2, and DT1-5. The structural composition and properties of these fractions were investigated. The predominant EPS, DT1-0, was identified as a novel heteropolysaccharide composed of fructose and glucose with branched structures. The repeating unit was determined to be [4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-β-D-Fruf-(2→6)-β-D-Fruf-(2→6-)-β-D-Fruf-(2→], with fructose and glucose connected by β-(2→1) and α-(1→4) glycosidic linkages between the third fructose and the first glucose, respectively. The molecular weight (Mw) was estimated to be 4.253 × 103 Da. DT1-0 presented a smooth and porous surface structure as observed through SEM and exhibited a water-holding capacity of 504 ± 5.3%, maximum thermal stability at 245 °C, and an oil-holding capacity of 387 ± 1.9% for coconut oil. DT1-2 was identified as a fructooligosaccharide. DT1-5 was characterized as a polysaccharide composed of glucose and fructose. In conclusion, these findings provide substantial support for the further application of B. subtilis strain DT1 and its EPS fractions, DT1-0, DT1-2, and DT1-5, as potential alternatives for functional food additives or ingredients.

An approach to manufacturing well-being milk chocolate in partial replacement of lecithin by the functional plant-based combination.

Lecithin is constituted of a glycerophospholipid mixture and is abundantly used as an emulsifying agent in various food applications including chocolate production. However, overconsumption of lecithin may create an adverse effect on human health. Thus, this study aims to replace the lecithin with plant-based gums. Different ratios of guar and arabic gum (25%-75%) and their blend (25%-75%) were employed as partial replacement of lecithin. Milk chocolate prepared using 40% guar gum (60GGL [guar gum, lecithin]), 25% arabic gum (75AGL [arabic gum, lecithin]), and a blend of 15 arabic gum and 10 guar gum (65AGGL [arabic gum, guar gum, lecithin]) showed similar rheological behavior as compared to control chocolate (100% lecithin). The fat content of 65AGGL (37.85%) was significantly lower than that of the control sample (43.37%). Rheological behavior exhibited shear-thinning behavior and samples (60GGL-75GGL-80GGL, 65AGL-75AGL, and 65AGGL-75AGGL) showed similar rheological properties as compared to control. The chocolate samples (60GGL and 65AGGL) showed significantly (p < .05) higher hardness values (86.01 and 83.55 N) than the control (79.95 N). As well, gum-added chocolates exhibited higher thermal stability up to 660°C as compared to the control sample. The Fourier transform infrared spectroscopy (FTIR) analysis revealed predominant β-(1 → 4) and β-(1 → 6) glycosidic linkages of the gums and lecithin. Sensory evaluation revealed a comparable score of gum-added milk chocolate in comparison to control samples in terms of taste, texture, color, and overall acceptance. Thus, plant exudate gums could be an excellent alternative to lecithin in milk chocolate, which can enhance the textural properties and shelf life.

Improvement of the catalytic performance of chitosanase Csn-PD from Paenibacillus dendritiformis by semi-rational design

Chitooligosaccharides (COS) possess versatile functional properties that have found extensive applications across various fields. Chitosanase can specifically hydrolyze β-1,4 glycosidic bonds in chitosan to produce COS. In this study, Csn-PD, a glycoside hydrolase family 46 chitosanase from Paenibacillus dendritiformis, which produces (GlcN)2 as its main product, was rationally redesigned aiming to improve its catalytic performance. Based on the results of molecular docking analysis and multiple sequence alignment, four amino acid residues in Csn-PD (I101, T120, T220, and Y259) were pinpointed for targeted mutations. Beneficial mutations in terms of enhanced catalytic activity were then combined by site-directed mutagenesis. Notably, the most promising variant, Csn-PDT6 (Csn-PD I101M/T120E/T220G), exhibited an impressive eight-fold surge in activity compared to the wild-type Csn-PD. This heightened enzymatic activity was complemented by an enhanced pH stability profile. A compelling feature of Csn-PDT6 is its preservation of the hydrolytic product profile observed in Csn-PD. This characteristic further accentuates its candidacy for the targeted production of (GlcN)2. The success of our strategic approach is vividly illustrated by the significant improvements achieved in the catalytic performance of the chitosanase, encompassing both its activity and stability. These developments offer a valuable model that may have implications for the semi-rational design of other enzymes.