Production Of Oligosaccharides Research Articles

Isolation of a novel Bacillus strain with industrial potential of producing alkaline chitosanase

A novel Bacillus strain, designated as HZ20-1, was discovered. This strain can produce naturally alkaline chitosanase without induction. Genomic analysis revealed that this chitosanase belongs to glycoside hydrolase family 8. When colloidal chitosan is used as a substrate, the maximum enzyme activity of 2.39 ± 0.03 U/mL is observed at a pH range of 8.5–9. This alkaline enzyme holds advantages over common acidic enzymes in various fields such as medicine, the environment, and daily chemical products, and thus has great market value. Moreover, as the chitosanase is a constitutive enzyme, there is no need for substrate induction. This can simplify fermentation equipment and reduce production cost. Additionally, in a preliminary study, we found that this strain can also degrade chitin and chitosan derivatives. After analysis, it was discovered that it has genes in glycoside hydrolase families 18 and 23. By controlling the enzymatic hydrolysis time, it is possible to produce products with different molecular weights, including N-acetylglucosamine, glucosamine, chito-oligosaccharides, and chitin oligosaccharides. Consequently, Bacillus sp. HZ20-1 is considered to have great potential for future industrial production of enzymes and oligosaccharides.

Preparation of carboxymethylated curdlan oligosaccharides and application on plant disease control

Curdlan is produced by fermentation of microorganisms, which is an insoluble β-(1 → 3)-D-glucans. To better effectively utilize native curdlan, firstly, a derivative from curdlan, carboxymethylated curdlan (CMCD), with different degrees of substitution (DS) DS ∼ 0.20, DS ∼ 0.43 and DS ∼ 0.82 were prepared in this study. Carboxymethylation increases solubility in water more than native curdlan. Moreover, CMCDs were investigated to be hydrolyzed by CcGluE, an endo-β-1 → 3-glucanase and generated the degradation products were oligosaccharides with degrees of polymerization (DP) mainly ranging from 2 to 7. CcGluE also showed high thermal and pH stability when CMCD ∼ 0.43 was used as a substrate. Then these oligosaccharides generated by different CMCDs were applied to Arabidopsis and the activity in inducing defense responses were detected after being treated by the pathogen of Pseudomonas syringae pv tomato DC3000 (Pst DC3000). CMCD (DS ∼ 0.20) degradation oligosaccharide (CMCD ∼ 0.20 OS) pre-treatment was the just one that significantly enhanced the disease resistance to Pst DC3000, which is mediated by the salicylic acid (SA) signaling pathway in plants. The findings offer new insights into the application of curdlan, demonstrating that carboxymethylation enhances its solubility in water. Additionally, the oligosaccharide products derived from CMCD degradation show promising prospects for controlling plant diseases in agriculture.

Enzymatic production, physicochemical characterization, and prebiotic potential of pectin oligosaccharides from pisco grape pomace

The prebiotic capacity of Pectin Oligosaccharides (POS) is influenced by structural factors such as molecular size, composition, and degree of esterification, which affect their interaction with the gut microbiota. While existing literature has predominantly examined POS derived from apple and citrus pectins, the extrapolation of these findings to other pectin sources remains complex due to variations in their composition. This study focused on obtaining POS with prebiotic potential from pisco grape pomace through controlled enzymatic hydrolysis, resulting in three molecular size fractions: <3 kDa, 3–10 kDa, and > 10 kDa. The POS fractions were analyzed using FTIR, HPSEC, HPLC, and MALDI-TOF-MS techniques to characterize their physical-chemical properties. Each fraction presented distinct compositions, with the <3 kDa fraction showing a higher concentration of galacturonic acid and glucose, while the >10 kDa fraction was also composed of rhamnose and arabinose. Notably, the <3 kDa fraction supported greater biomass growth of the probiotic strain Lactobacillus casei ATCC 393 compared to the other fractions. In contrast, the non-probiotic strain Escherichia coli ATCC 25922 achieved the lowest biomass with this fraction. Consequently, the <3 kDa POS fraction exhibited the highest prebiotic index. This fraction, composed of oligomers from the rhamnogalacturonan region and arabino-oligosaccharides with a degree of polymerization between two and five, highlights its potential for further research and applications. Therefore, investigating other sources and optimizing extraction conditions could lead to developing novel prebiotic formulations that supply specific probiotic strains for a symbiotic product.

Low molecular weight carbohydrates and abiotic stress tolerance in lentil (Lens culinaris Medikus): a review.

Lentil (Lens culinaris Medikus) is a nutrient-rich, cool-season food legume that is high in protein, prebiotic carbohydrates, vitamins, and minerals. It is a staple food in many parts of the world, but crop performance is threatened by climate change, where increased temperatures and less predictable precipitation can reduce yield and nutritional quality. One mechanism that many plant species use to mitigate heat and drought stress is the production of disaccharides, oligosaccharides and sugar alcohols, collectively referred to as low molecular weight carbohydrates (LMWCs). Recent evidence indicates that lentil may also employ this mechanism - especially raffinose family oligosaccharides and sugar alcohols - and that these may be suitable targets for genomic-assisted breeding to improve crop tolerance to heat and drought stress. While the genes responsible for LMWC biosynthesis in lentil have not been fully elucidated, single nucleotide polymorphisms and putative genes underlying biosynthesis of LMWCs have been identified. Yet, more work is needed to confirm gene identity, function, and response to abiotic stress. This review i) summarizes the diverse evidence for how LMWCs are utilized to improve abiotic stress tolerance, ii) highlights current knowledge of genes that control LMWC biosynthesis in lentil, and iii) explores how LMWCs can be targeted using diverse genomic resources and markers to accelerate lentil breeding efforts for improved stress tolerance.

Identification and Characterization of a Highly Active Hyaluronan Lyase from Enterobacter asburiae.

Hyaluronic acid (HA) is a well-known functional marine polysaccharide. The utilization and derivative development of HA are of great interest. Hyaluronan lyase has wide application prospects in the production of HA oligosaccharides and lower molecular weight HA. In this study, a strain of Enterobacter asburiae CGJ001 with high hyaluronan lyase activity was screened from industrial wastewater. This strain exhibited an impressive enzyme activity of 40,576 U/mL after being incubated for 14 h. Whole genome sequencing analysis revealed that E. asburiae CGJ001 contained a cluster of genes involved in HA degradation, transport, and metabolism. A newly identified enzyme responsible for glycosaminoglycan degradation was designated as HylEP0006. A strain of E. coli BL21(DE3)/pET-22b(+)-hylEP0006 was successfully constructed. HylEP0006 exhibited optimal degradation at 40 °C and pH 7.0, showing a high activity of 950,168.3 U/mg. HylEP0006 showed specific activity against HA. The minimum degradation fragment of HylEP0006 was hyaluronan tetrasaccharides, and HylEP0006 could efficiently degrade HA into unsaturated disaccharides (HA2), with HA2 as the final product. These characteristics indicate that HylEP0006 has a potential application prospect for the extraction and utilization of hyaluronic acid.

Enhanced algin oligosaccharide production through selective breeding and optimization of growth and degradation conditions in Cobetia sp. cqz5-12-M1

Algin oligosaccharides have been applied in diverse industries and could be innovative synthesized by alginate-degrading bacteria. For enhance the alginate degradation efficiency to produce more algin oligosaccharides, a mutant strain (Cobetia sp. cqz5-12-M1) was obtained through the complex mutagenesis using UV and the alkylating agent 1-methyl-3-nitro-1-nitrosoguanidine. The enzyme activity of the fermentation supernatant of mutant exhibited a significant 38.09% (53.98 ± 0.69 U/mL) increase, and its optimal growth conditions were determined as: 5 g/L sodium alginate, 5 g/L yeast powder, 30 g/L NaCl, 2 g/L K2HPO4, 2 g/L KH2PO4, 1 g/L MgSO4•7H2O, 0.01 g/L FeSO4•7H2O, pH 6.5, and 34 ℃. Moreover, its optimal degradation conditions were identified as: 5 g/L sodium alginate, 5 g/L yeast powder, 30 g/L NaCl, 2 g/L K2HPO4, 2 g/L KH2PO4, 1 g/L MgSO4•7H2O, 0.01 g/L FeSO4•7H2O, pH 6.5, 31 ℃ and 72 h, yielding an enzyme activity of 120.98 ± 1.40 U/mL in the fermentation supernatant. Conclusive experiments on reagent tolerance revealed the growth of the mutant strain was significantly inhibited by 3% hydrogen peroxide, 5% carbolic acid, and 10 mg/mL gatifloxacin. Additionally, the alginate degradation capacity of mutant strain was highly significantly inhibited by 75% ethanol and all tested antibiotics.

Chemical and enzymatic hydrolysis of alginate: a review

Abstract Alginate is a brown seaweed-based linear polysaccharide of D – mannuronic acid and L – guluronic acid residues. Hydrolysis products of the polysaccharide, specifically oligosaccharides, have been receiving increasing interest, due to their significant bioactivity and potential utilisation routes. The bioactivity of alginate oligosaccharides is closely linked to structural characteristics, namely: molecular weight, degree of polymerisation, and ratio of the monomers (the M/G ratio). Hence, potential applications (such as utilisation as a biostimulant fertilizer) depend on these parameters. This review focuses on recent advances in producing alginate oligosaccharides using chemical or enzymatic methods. The literature survey includes utilisation of these methods at both laboratory and industrial scale. For the chemical methods, we assessed the standard laboratory scale procedures of alginate oligosaccharide production, the potential of scaling up to an industrial level, and the subsequent challenges. For the enzymatic route, we provide an overview of alginate lyases and the application perspectives of enzymatic hydrolysis of alginate.

Integrated enzymatic hydrolysis of crude red onion extract and yeast treatment for production and purification of short-chain inulin and inulin neoseries oligosaccharides

As has been confirmed in our previous study, red onion is a promising source of inulin-fructan, inulin neoseries fructan, and their fructooligosaccharides (FOSs). Short-chain FOSs (SCFOSs) are potential prebiotics as they provide many health promoting benefits. However, short-chain inulin neoseries oligosaccharides isolated from plants have scarcely been studied. After prebiotic production, a purification step is essential to produce a functional prebiotic. The aim of this study was to develop new processes of integrated enzymatic hydrolysis of crude red onion extract and yeast treatment for production and purification of SCFOSs. Endo-inulinase is a key enzyme for hydrolysis of crude red onion extract to produce SCFOSs, while Candida orthopsilosis FLA44.2 was employed for purification of the produced SCFOSs. Three different strategies, including two-step (TSP), simultaneous (SP), and semi-simultaneous (SSP) production processes, were designed and investigated as potentially simple, cost-effective, and timesaving production processes. The SP and SSP processes met the objective criteria by exhibiting desirable yields of SCFOSs and the optimal purity of SCFOSs when compared with the TSP process. Notably, the SP process was highlighted as it was simpler than the SSP process. Under optimal conditions (0.4 U of endo-inulinase/g total fructans, 5 % v/v of yeast inoculum and reaction time of 72 h), the SP process conducted in 1-dm3 flasks produced SCFOSs of 87 g/dm3 with neo-GF2 as the main constituent (42 g/dm3) and a purity of 100 %. The ratio of total SCFOSs to total fructans was 0.93 indicating that fructans of the red onion extract have been transformed to SCFOSs.

Guide RNA structure design enables combinatorial CRISPRa programs for biosynthetic profiling

Engineering metabolism to efficiently produce chemicals from multi-step pathways requires optimizing multi-gene expression programs to achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important tools for programming multi-gene expression, but poor predictability of guide RNA folding can disrupt expression control. Here, we correlate efficacy of modified guide RNAs (scRNAs) for CRISPR activation (CRISPRa) in E. coli with a computational kinetic parameter describing scRNA folding rate into the active structure (rS = 0.8). This parameter also enables forward design of scRNAs, allowing us to design a system of three synthetic CRISPRa promoters that can orthogonally activate (>35-fold) expression of chosen outputs. Through combinatorial activation tuning, we profile a three-dimensional design space expressing two different biosynthetic pathways, demonstrating variable production of pteridine and human milk oligosaccharide products. This RNA design approach aids combinatorial optimization of metabolic pathways and may accelerate routine design of effective multi-gene regulation programs in bacterial hosts.

The multi-modal sensory effect on sour taste perception during oral processing of starch-thickened fluids

Sour taste is one of the fundamental taste attributes and considered as a saliva secretion stimulant facilitating consumers with xerostomia and swallowing difficulties. The dynamic sour taste perception during oral processing of starch-thickened fluid was therefore investigated in this study. Recruited healthy subjects were divided into those with high (H_AMS) and low (L_AMS) salivary α-amylase activity groups and their temporal dominant sensory evaluation were analyzed while oral processing starch-thickened fluid samples with different concentration of citric acid. By simultaneously evaluating the physical and chemical properties of subjects' expectorated fluid/saliva mixtures, the complex and multi-modal interplay between fluid viscosity (tactile perception), citric acid concentration (taste perception), salivary α-amylase activity (binary taste interaction) was successfully demonstrated. The sour taste perception was found to be greatly affected by the continuous starch-thickened fluid hydrolysis by salivary α-amylase, resulting in a gradual production of sweet-tasting oligosaccharides and simultaneous decrease in fluid viscosity, and group variations (H_AMS vs. L_AMS) were pronouncedly observed in the hydrolyzed oligosaccharide concentrations (glucose, maltose, and maltotriose) which suitably corresponded to the selected dominant sensory attributes including perceived taste differences and taste dominant duration. These findings suggest that salivary biochemical properties and the thickened fluid type greatly influence consumers’ sensory perception. This could provide valuable insights for designing and optimizing thickened foods and beverages that are palatable in sensory properties and meet the needs of special consumers.

Enhancing the rigidity of the catalytic pocket entrance to improve catalytic efficiency and thermal stability of alginate Lyase from Pseudoalteromonas

Alginate lyase plays a critical role in the production of alginate oligosaccharides (AOS), necessitating efficient activity and a certain level of thermal stability for industrial AOS production. In this study, through sequence alignment and structural analysis, a non-catalytic amino acid residue, Ala195, located at the entrance of the catalytic pocket was identified. This residue is crucial for the catalytic efficiency and thermal stability of Alg2944, a member of the polysaccharide lyase family 18. The specific activities of A195F, A195I, and A195L were enhanced by 1.84-, 1.39-, and 1.98-fold, respectively, while the thermal stability of mutants A195L and A195I showed significant improvements at 35 °C and 40 °C compared to the wild-type (WT) enzyme. Furthermore, molecular docking, structural analysis, and molecular dynamics (MD) simulations revealed that the enhanced specific activity and thermal stability of the A195L mutant can be attributed to the creation of a hydrophobic pocket, increased rigidity in the loop region, and a notably lower binding free energy of the A195L mutant (-45.96 ± 7.69 kcal/mol) compared to the WT (-38.40 ± 4.25 kcal/mol). These findings underscore the essential role of rigidity and hydrophobicity at the entrance of the catalytic pocket in ensuring both catalytic efficiency and thermal stability of alginate lyase.

Improving Agar Degradation Activity of Vibrio natriegens WPAGA4 via Atmospheric and Room Temperature Plasma (ARTP)

Agar oligosaccharides from the degradation of agar harbor great potential in the food and pharmaceutical industries. An agar-degrading bacterium, Vibrio natriegens WPAGA4, was isolated from the deep sea in our previous work. However, the agar-degrading activity of WPAGA4 remains to be improved for more production benefits of this strain. The aim of this study was to enhance the agar-degrading activity of WPAGA4 by using atmospheric and room temperature plasma (ARTP) mutagenesis. Three mutant strains, including T1, T2, and T3, with good genetic stability were obtained, and the agar-degrading activities of these strains increased by 136%, 141%, and 135%, respectively. The optimal temperature and pH for agar degradation were slightly changed in the mutant strains. No sequence mutation was detected in all the agarase genes of WPAGA4, including agaW3418, agaW3419, agaW3420, and agaW3472. However, ARPT mutagenesis increased the relative expression levels of agaW3418, agaW3419, and agaW3420 in the mutant strains, which could be the reason for the improvement of degradation activities in the mutant strains. Furthermore, T3 had the lowest consumption rate of agar oligosaccharide, which was 21% less than the wild-type strain. Therefore, T3 possessed a preferable production value due to its higher degrading activity and lower consumption of agar oligosaccharides. The current work enhanced the agar-degrading activity of WPAGA4 and offered strains with greater potential for agar oligosaccharide production, thereby laying the foundation for industrial applications.

Heterologous Expression and Characterization of a pH-Stable Chitinase from Micromonospora aurantiaca with a Potential Application in Chitin Degradation.

To promote the bioconversion of marine chitin waste into value-added products, we expressed a novel pH-stable Micromonospora aurantiaca-derived chitinase, MaChi1, in Escherichia coli and subsequently purified, characterized, and evaluated it for its chitin-converting capacity. Our results indicated that MaChi1 is of the glycoside hydrolase (GH) family 18 with a molecular weight of approximately 57 kDa, consisting of a GH18 catalytic domain and a cellulose-binding domain. We recorded its optimal activity at pH 5.0 and 55 °C. It exhibited excellent stability in a wide pH range of 3.0-10.0. Mg2+ (5 mM), and dithiothreitol (10 mM) significantly promoted MaChi1 activity. MaChi1 exhibited broad substrate specificity and hydrolyzed chitin, chitosan, cellulose, soluble starch, and N-acetyl chitooligosaccharides with polymerization degrees ranging from three to six. Moreover, MaChi1 exhibited an endo-type cleavage pattern, and it could efficiently convert colloidal chitin into N-acetyl-D-glucosamine (GlcNAc) and (GlcNAc)2 with yields of 227.2 and 505.9 mg/g chitin, respectively. Its high chitin-degrading capacity and exceptional pH tolerance makes it a promising tool with potential applications in chitin waste treatment and bioactive oligosaccharide production.

Arabinoxylo-oligosaccharides production from unexploited agro-industrial sesame (Sesamum indicum L.) hulls waste

This work demonstrates that sesame (Sesamum indicum L.) hull, an unexploited food industrial waste, can be used as an efficient source for the extraction of hemicellulose and/or pectin polysaccharides to further obtain functional oligosaccharides. Different polysaccharides extraction methods were surveyed including alkaline and several enzymatic treatments. Based on the enzymatic release of xylose, arabinose, glucose, and galacturonic acid from sesame hull by using different enzymes, Celluclast®1.5 L, Pectinex®Ultra SP-L, and a combination of them were selected for the enzymatic extraction of polysaccharides at 50 °C, pH 5 up to 24 h. Once the polysaccharides were extracted, Ultraflo®L was selected to produce arabinoxylo-oligosaccharides (AXOS) at 40 °C up to 24 h. Apart from oligosaccharides production from extracted polysaccharides, alternative approaches for obtaining oligosaccharides were also explored. These were based on the analysis of the supernatants resulting from the polysaccharide extraction, alongside a sequential hydrolysis performed with Celluclast®1.5 L and Ultraflo®L of the starting raw sesame hull. The different fractions obtained were comprehensively characterized by determining low molecular weight carbohydrates and monomeric compositions, average Mw and dispersity, and oligosaccharide structure by MALDI-TOF-MS. The results indicated that sesame hull can be a useful source for polysaccharides extraction (pectin and hemicellulose) and derived oligosaccharides, especially AXOS.

Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates which support the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the approximately 200 structurally diverse HMOs at scale has proved difficult. Here we produce a diversity of HMOs by leveraging the robust carbohydrate anabolism of plants. This diversity includes high-value and complex HMOs, such as lacto-N-fucopentaose I. HMOs produced in transgenic plants provided strong bifidogenic properties, indicating their ability to serve as a prebiotic supplement with potential applications in adult and infant health. Technoeconomic analyses demonstrate that producing HMOs in plants provides a path to the large-scale production of specific HMOs at lower prices than microbial production platforms. Our work demonstrates the promise in leveraging plants for the low-cost and sustainable production of HMOs.

Significantly improving the thermal stability of alginate lyase AlyC3 from Psychromonas sp. C-3 by computational redesign

Alginate lyase is a key enzyme in the bioproduction of alginate oligosaccharides. The alginate lyase AlyC3, derived from Psychromonas sp. C-3, can hydrolyze homopolymerized mannuronic acid into oligosaccharides (primarily composed of mannuronate trimer), which possess physiological properties such as bacteriostatic and antioxidant activities and show a better application prospect. However, the poor thermal stability hinders its potential application in industrial production. In this study, AlyC3 mutants A35P, L95F, N138Y and M162I were obtained through the protein repair one-stop shop (PROSS) design combined with protein data bank in Europe (proteins, interfaces, structures and assemblies) (PDBePISA) analysis of the dimer interface. The results demonstrated that the thermodynamic and kinetic stability of these single-point mutants were significantly improved compared to the wild-type (WT), with the melting temperature increased by 4.33, 9.35, 9.74 and 11.24 °C, respectively, while the half-life (t1/2) was 1.66, 1.02, 1.02 and 1.53 times higher than that of WT, respectively. Based on this, the beneficial mutations were further combined to obtain a combinatorial mutant, A35P-L95F–N138Y-M162I, which exhibited the superior thermal stability. Notably, the combinatorial mutant's melting and optimal temperature increased by 17.05 and 10 °C, respectively; moreover, the mutant enzyme's t1/2 and enzyme activity were 8.21 times and 123.93% higher than those of the WT, respectively. Structural analyses indicated that increased hydrophobic interactions and polar bonding between the mutated and nearby residues, with improved rigidity in the flexible loop, might account for the significantly improved thermal stability of the reengineered AlyC3, which makes it a robust candidate for alginate oligosaccharide production.

The culture of Chlorella with livestock wastewater and the application of its oligosaccharides in promoting rice seed germination under high salinity conditions

Algal oligosaccharides, derived from polysaccharides in algal biomass, exhibit bioactive properties that enhance plant antioxidant enzyme activity and free radical scavenging. Currently, algal oligosaccharides are mainly produced from large algae, while studies on preparing algal oligosaccharides from microalgae are relatively insufficient. This study focuses on utilizing Chlorella cultivated under controlled conditions using diluted livestock wastewater to produce algal oligosaccharides and investigates their impact on rice seed germination under salt stress. Results show that culturing Chlorella with diluted livestock wastewater not only stimulated algal growth but also efficiently removed nitrogen, phosphorus, and other nutrients. Algal oligosaccharides demonstrated significant improvements in rice seed germination, vigor indices, biomass accumulation, and nitrogen absorption under salt stress. Physiological indicators revealed elevated osmotic adjustment substances, increased chlorophyll content, and alleviated oxidative damage in rice seedlings treated with algal oligosaccharides. The study introduces an innovative method of using livestock wastewater for algal oligosaccharides production, emphasizing its dual benefits including efficient resource utilization and significant cost reduction. The findings also support the theoretical application of Chlorella-derived algal oligosaccharides in enhancing rice cultivation under salt stress.

Pichia pastoris Mediated Digestion of Water-Soluble Polysaccharides from Cress Seed Mucilage Produces Potent Antidiabetic Oligosaccharides.

Diabetes mellitus is a heterogeneous metabolic disorder that poses significant health and economic challenges across the globe. Polysaccharides, found abundantly in edible plants, hold promise for managing diabetes by reducing blood glucose levels (BGL) and insulin resistance. However, most of these polysaccharides cannot be digested or absorbed directly by the human body. Here we report the production of antidiabetic oligosaccharides from cress seed mucilage polysaccharides using yeast fermentation. The water-soluble polysaccharides extracted from cress seed mucilage were precipitated using 75% ethanol and fermented with Pichia pastoris for different time intervals. The digested saccharides were fractionated through gel permeation chromatography using a Bio Gel P-10 column. Structural analysis of the oligosaccharide fractions revealed the presence of galacturonic acid, rhamnose, glucuronic acid, glucose and arabinose. Oligosaccharide fractions exhibited the potential to inhibit α-amylase and α-glucosidase enzymes in a dose-dependent manner in vitro. The fraction DF73 exhibited strong inhibitory activity against α-amylase with IC50 values of 38.2 ± 1.12 µg/mL, compared to the positive control, acarbose, having an IC50 value of 29.18 ± 1.76 µg/mL. Similarly, DF72 and DF73 showed the highest inhibition of α-glucosidase, with IC50 values of 9.26 ± 2.68 and 50.47 ± 5.18 µg/mL, respectively. In in vivo assays in streptozotocin (STZ)-induced diabetic mice, these oligosaccharides significantly reduced BGL and improved lipid profiles compared to the reference drug metformin. Histopathological observations of mouse livers indicated the cytoprotective effects of these sugars. Taken together, our results suggest that oligosaccharides produced through microbial digestion of polysaccharides extracted from cress seed mucilage have the potential to reduce blood glucose levels, possibly through inhibition of carbohydrate-digesting enzymes and regulation of the various signaling pathways.

Structural insights into starch-metabolizing enzymes and their applications.

Starch is a polysaccharide produced exclusively through photosynthesis in plants and algae; however, is utilized as an energy source by most organisms, from microorganisms to higher organisms. In mammals and the germinating seeds of plants, starch is metabolized by simple hydrolysis pathways. Moreover, starch metabolic pathways via unique oligosaccharides have been discovered in some bacteria. Each organism has evolved enzymes responsible for starch metabolism that are diverse in their enzymatic properties. This review, focusing on eukaryotic α-glucosidases and bacterial α-glucoside-hydrolyzing enzymes, summarizes the structural aspects of starch-metabolizing enzymes belonging to glycoside hydrolase families 15, 31, and 77 and their application for oligosaccharide production.

Combinatorial metabolic engineering of Escherichia coli for de novo production of structurally defined and homogeneous Amino oligosaccharides

Amino oligosaccharides (AOs) possess various biological activities and are valuable in the pharmaceutical, food industries, and agriculture. However, the industrial manufacturing of AOs has not been realized yet, despite reports on physical, chemical, and biological approaches. In this study, the de novo production of chitin oligosaccharides (CHOS), a type of structurally defined AOs, was achieved in Escherichia coli through combinatorial pathway engineering. The most suitable glycosyltransferase for CHOS production was found to be NodCL from Mesorhizobium Loti. Then, by knocking out the nagB gene to block the flow of N-acetyl-d-glucosamine (NAG) to the glycolytic pathway in E. coli and adjusting the copy number of NodCL-coding gene, the CHOS yield was increased by 6.56 times. Subsequently, by introducing of UDP-N-acetylglucosamine (UDP-GlcNAc) salvage pathway for and optimizing fermentation conditions, the yield of CHOS reached 207.1 and 468.6 mg/L in shake-flask cultivation and a 5-L fed-batch bioreactor, respectively. Meanwhile, the concentration of UDP-GlcNAc was 91.0 mg/L, the highest level reported in E. coli so far. This study demonstrated, for the first time, the production of CHOS with distinct structures in plasmid-free E. coli, laying the groundwork for the biosynthesis of CHOS and providing a starting point for further engineering and commercial production.