D-allulose 3-epimerase for low-calorie D-allulose synthesis: microbial production, characterization, and applications.

D-allulose, an epimer of D-fructose at C-3 position, is a low-calorie rare sugar with favorable physiochemical properties and special physiological functions, which displays promising perspectives in the food and pharmaceutical industries. Currently, D-allulose is extremely sparse in nature and is predominantly biosynthesized through the isomerization of D-fructose by D-allulose 3-epimerase (DAEase). In recent years, D-allulose 3-epimerase as the key biocatalyst for D-allulose production has received increasing interest. The current review begins by providing a summary of D-allulose regarding its characteristics and applications, as well as different synthesis pathways dominated by biotransformation. Then, the research advances of D-allulose 3-epimerase are systematically reviewed, focusing on heterologous expression and biochemical characterization, crystal structure and molecular modification, and application in D-allulose production. Concerning the constraint of low yield of DAEase for industrial application, this review addresses the various attempts made to promote the production of DAEase in different expression systems. Also, various strategies have been adopted to improve its thermotolerance and catalytic activity, which is mainly based on the structure-function relationship of DAEase. The application of DAEase in D-allulose biosynthesis from D-fructose or low-cost feedstocks through single- or multi-enzymatic cascade reaction has been discussed. Finally, the prospects for related research of D-allulose 3-epimerase are also proposed, facilitating the industrialization of DAEase and more efficient and economical bioproduction of D-allulose.

Study on the gelatinization and digestive characteristics of wheat starch and potato starch under low moisture conditions.

This study explored the gelatinization and digestive characteristics of wheat and potato starches under low moisture conditions using identical processing parameters. The results revealed that potato starch exhibited greater resistance to digestion than wheat starch, with an enzyme hydrolysis rate 18 % to 30 % lower than wheat starch under the same conditions. The analysis of particle size, swelling power, and low-field NMR demonstrated that potato starch required almost 40 % more moisture for full gelatinization than wheat starch, indicating that low-moisture conditions could not meet the significant water demand of potato starch. Additionally, the DSC analysis showed that potato starch had superior thermal stability, with To of 62.13 °C and ΔH of 16.30 (J/g). Subsequently, the microscopy results showed that the partially gelatinized wheat starch had a rough, porous surface, allowing enzymes for direct access to hydrolysis. In contrast, the potato starch had smoother and less damaged particles without visible pores, enzymes had to degrade it progressively, layer by layer. Furthermore, potato starch still exhibited a lower enzyme hydrolysis rate than wheat starch under the same gelatinization levels. Overall, potato starch is more resistant to hydrolysis and gelatinization in low-moisture environments, making potato starch suitable for low-digestibility products like potato biscuits or chips.

Enzymatic modification lowers syneresis in corn starch gels during freeze-thaw cycles through 1,4-α-glucan branching enzyme.

The current research in the food industry regarding enzymatic modification to enhance the freeze-thaw (FT) stability of starch is limited. The present study aimed to investigate the FT stability of normal corn starch (NCS) modified using 1,4-α-glucan branching enzyme (GBE) derived from Geobacillus thermoglucosidans STB02. Comprehensive analyses, including syneresis, scanning electron microscopy, and low-field nuclear magnetic resonance, collectively demonstrated the enhanced FT stability of GBE-modified corn starch (GT-NCS-30) in comparison to its native form. Its syneresis was 66.4 % lower than that of NCS after three FT cycles. Notably, GBE treatment induced changes in the pasting properties and thermal resistance of corn starch, while simultaneously enhancing the mechanical strength of the starch gel. Moreover, X-ray diffractograms and microstructural assessments of freeze-thawed gels indicated that GBE treatment effectively hindered the association of corn starch molecules, particularly amylose retrogradation. The enhanced FT stability of GBE-modified starch can be attributed to alterations in the starch structure induced by GBE. This investigation establishes a foundation for further exploration into the influence of GBE treatment on the FT stability of starch and provides a theoretical basis for further research in this area.

Carbohydrate binding modules: Compact yet potent accessories in the specific substrate binding and performance evolution of carbohydrate-active enzymes.

Carbohydrate binding modules (CBMs) are independent non-catalytic domains widely found in carbohydrate-active enzymes (CAZymes), and they play an essential role in the substrate binding process of CAZymes by guiding the appended catalytic modules to the target substrates. Owing to their precise recognition and selective affinity for different substrates, CBMs have received increasing research attention over the past few decades. To date, CBMs from different origins have formed a large number of families that show a variety of substrate types, structural features, and ligand recognition mechanisms. Moreover, through the modification of specific sites of CBMs and the fusion of heterologous CBMs with catalytic domains, improved enzymatic properties and catalytic patterns of numerous CAZymes have been achieved. Based on cutting-edge technologies in computational biology, gene editing, and protein engineering, CBMs as auxiliary components have become portable and efficient tools for the evolution and application of CAZymes. With the aim to provide a theoretical reference for the functional research, rational design, and targeted utilization of novel CBMs in the future, we systematically reviewed the function-related characteristics and potentials of CAZyme-derived CBMs in this review, including substrate recognition and binding mechanisms, non-catalytic contributions to enzyme performances, module modifications, and innovative applications in various fields.

Effects of hydrocolloids on mechanical properties, viscoelastic and microstructural properties of starch-based modeling clay.

The effects of various hydrocolloids (guar gum, xanthan gum, and carboxymethyl cellulose) on the texture, rheology, and microstructural properties of modeling clay prepared with cassava starch were investigated. Notably, incorporation of 3 % guar gum and 4 % xanthan gum into starch-based modeling clay resulted in enhancements of 94.12 % and 77.47 % in cohesiveness, and 64.70 % and 66.20 % in extensibility, respectively. For starch-based modeling clay with added guar gum and xanthan gum, compared to formulations without hydrocolloids, the linear viscoelastic range exceeded 0.04 %, and the frequency dependence of both maximum creep compliance (Jmax) and storage modulus (G') was significantly reduced. This indicates a more stable network structure and enhanced resistance to deformation. Results from Fourier Transform Infrared (FTIR) spectroscopy and X-ray diffraction (XRD) confirmed that the physical interactions between starch and various hydrocolloids, along with the addition of these hydrocolloids, inhibited the degradation effect of thermomechanical processing on the crystalline structure of starch. With the addition of guar gum, it is observed that a continuous and dense network structure forms within the starch-based modeling clay, and starch particles are distributed uniformly. In conclusion, hydrocolloids enhances the properties of starch-based modeling clay, introducing an innovative solution to the modeling clay sector.

Pullulanase pretreatment of highly concentrated maltodextrin solution improves maltose yield during ß-amylase-catalyzed saccharification.

Increasing the substrate concentration can effectively reduce energy consumption and result in more economic benefits in the industrial production of maltose, but this process remarkably increases the viscosity, which has a negative effect on saccharification. To improve saccharification efficiency, pullulanase is usually employed. In the conventional process of maltose production, pullulanase is added at the same time with ß-amylase or later, but this process seems inefficient when the substrate concentration is high. Herein, a novel method was introduced to enhance the maltose yield under high substrate concentration. The results indicated that the pullulanase pretreatment of highly concentrated maltodextrin solution for 2 h greatly affects the final conversion rate of ß-amylase-catalyzed saccharification. The maltose yield reached 80.95 %, which is 11.8 % above the control value. Further examination confirmed that pullulanase pretreatment decreased the number of branch points of maltodextrin and resulted in a high content of oligosaccharides. These linear chains were suitable for ß-amylase-catalyzed saccharification to produce maltose. This research offers a new effective and green strategy for starch sugar production.

All-In-One OsciDrop Digital PCR System for Automated and Highly Multiplexed Molecular Diagnostics.

Digital PCR (dPCR) holds immense potential for precisely detecting nucleic acid markers essential for personalized medicine. However, its broader application is hindered by high consumable costs, complex procedures, and restricted multiplexing capabilities. To address these challenges, an all-in-one dPCR system is introduced that eliminates the need for microfabricated chips, offering fully automated operations and enhanced multiplexing capabilities. Using this innovative oscillation-induced droplet generation technique, OsciDrop, this system supports a comprehensive dPCR workflow, including precise liquid handling, pipette-based droplet printing, in situ thermocycling, multicolor fluorescence imaging, and machine learning-driven analysis. The system's reliability is demonstrated by quantifying reference materials and evaluating HER2 copy number variation in breast cancer. Its multiplexing capability is showcased with a quadruplex dPCR assay that detects key EGFR mutations, including 19Del, L858R, and T790M in lung cancer. Moreover, the digital stepwise melting analysis (dSMA) technique is introduced, enabling high-multiplex profiling of seven major EGFR variants spanning 35 subtypes. This innovative dPCR system presents a cost-effective and versatile alternative, overcoming existing limitations and paving the way for transformative advances in precision diagnostics.

Enhancement of ß-Cyclodextrin Production Using a Glycogen Debranching Enzyme from Saccharolobus solfataricus STB09.

Efficient production of cyclodextrins (CDs) has always been challenging. CDs are primarily produced from starch via cyclodextrin glycosyltransferase (CGTase), which acts on α-1,4 glucosidic bonds; however, α-1,6 glucosidic bonds in starch suppress the enzymatic production of CDs. In this study, a glycogen debranching enzyme from Saccharolobus solfataricus STB09 (SsGDE) was utilized to promote the production of ß-CD by hydrolyzing α-1,6 glucosidic bonds. The addition of SsGDE (750 U/g of starch) at the liquefaction stage remarkably improved the ß-CD yield, with a 43.9% increase. Further mechanism exploration revealed that SsGDE addition could hydrolyze specific branches with less generation of byproducts, thereby promoting CD production. The chain segments of a degree of polymerization ≥13 produced by SsGDE debranching could also be utilized by ß-CGTase to convert into CDs. Overall, these findings proposed a new approach of combining SsGDE with ß-CGTase to enhance the CD yield.

Effect of hydrocolloids on starch digestion: A review.

Rapidly digestible starch can increase postprandial blood sugar rapidly, which can be overcome by hydrocolloids. The paper aims to review the effect of hydrocolloids on starch digestion. Hydrocolloids used to reduce starch digestibility are mostly polysaccharides like xanthan gum, pectin, ß-glucan, and konjac glucomannan. Their effectiveness is related to their source and structure, mixing mode of hydrocolloid/starch, physical treatment, and starch processing. The mechanisms of hydrocolloid action include increased system viscosity, inhibition of enzymatic activity, and reduced starch accessibility to enzymes. Reduced starch accessibility to enzymes involves physical barrier and structural orderliness. In the future, physical treatments and intensity used for stabilizing hydrocolloid/starch complex, risks associated with different doses of hydrocolloids, and the development of related clinical trials should be focused on. Besides, investigating the effect of hydrocolloids on starch should be conducted in the context of practical commercial applications rather than limited to the laboratory level.

Optimization and analysis of dual-enzymatic synthesis for the production of linear glucan.

Linear α-glucan (LG), a linear polymer linked by α-1,4 bonds, has received increasing attention for its potential applications in synthetic polymer production. Notably, the functionality of LG is strongly influenced by its degree of polymerization (DP). In this study, SP and GP were successfully constructed and expressed. The reaction of enzymatic co-polymerization into LG was investigated. The preferred reaction was carried out at 37 °C and pH 7.4 for 72 h, with a maximum conversion rate of 25 %. Afterwards, two approaches were used to modulate the molecular structures of LGs. Firstly, LGs with distinct molecular weights ranging from 1062.33 ± 16.04 g/mol to 5679 ± 80.29 g/mol were obtained by adjusting the substrate/primer ratio during the LG synthesis process. Secondly, two distinct products could be produced by altering the enzyme addition method: short-chain LG with a DP < 10 (64.34 ± 0.54 %) or long-chain LG with a DP > 45 (45.57 ± 2.18 %). Additionally, theoretical synthesis model was constructed which subdivided the reaction into three stages to evaluate this dual-enzyme cooperative system. These findings have significant implications in promoting the application of LG in the fields of biomedicine and material science.

Effect of solution on starch structure: New separation approach of amylopectin fraction from gelatinized native corn starch.

The complete dissolution of starch without degradation are necessary prerequisites for starch fractionation to obtain amylose or amylopectin (AP). With the recent, continuous progress in finding efficient and eco-friendly starch-dissolving solutions, applying new solvents for starch fractionation is important. In this study, the effects of dimethyl sulfoxide (DMSO), NaOH, and CaCl2 solutions on starch structure and AP product parameters during starch fractionation were compared with respect to the starch deconstruction effect. This study proved that the CaCl2 solution could effectively dissolve corn starch (50 °C, solubility of 98.96 %), and promote the regeneration of starch into uniform and fine particles. Furthermore, the three solvents (DMSO, NaOH, and CaCl2) changed the crystal structure of corn starch, but they were all non-derivatizing solvents. The effect of the CaCl2 solution on the molecular structure of corn starch was the least significant of the three solvents. Finally, the extraction rate of AP from the CaCl2 solution reached 69.45 %. In conclusion, this study presents a novel and effective method for AP extraction.

The substitution sites of hydroxyl and galloyl groups determine the inhibitory activity of human pancreatic α-amylase in twelve tea polyphenol monomers.

Tea polyphenols have been reported as potential α-amylase inhibitors. However, the quantitative structure-activity relationship (QSAR) between tea polyphenols and human pancreas α-amylase (HPA) is not well understood. Herein, the inhibitory effect of twelve tea polyphenol monomers on HPA was investigated in terms of inhibitory activity, as well as QSAR analysis and interaction mechanism. The results revealed that the HPA inhibitory activity of theaflavins (TFs), especially theaflavin-3'-gallate (TF-3'-G, IC50: 0.313 mg/mL), was much stronger than that of catechins (IC50: 18.387-458.932 mg/mL). The QSAR analysis demonstrated that the determinant for the inhibitory activity of HPA was not the number of hydroxyl and galloyl groups in tea polyphenol monomers, while the substitution sites of these groups potentially might play a more important role in modulating the inhibitory activity. The inhibition kinetics and molecular docking revealed that TF-3'-G as a mixed-type inhibitor had the lowest inhibition constant and bound to the active sites of HPA with the lowest binding energy (-7.74 kcal/mol). These findings could provide valuable insights into the structures-activity relationships between tea polyphenols and the HPA inhibitors.

Effect of non-starch components on the structural properties, physicochemical properties and in vitro digestibility of waxy highland barley starch.

Highland barley (HB) endosperm with an amylose content of 0-10 % is called waxy HB (WHB). WHB is a naturally slow-digesting grain, and the interaction between its endogenous non-starch composition and the WHB starch (WHBS) has an important effect on starch digestion. This paper focuses on the mechanisms by which the components of ß-glucan, proteins and lipids affect the molecular, granular, crystalline structure and digestive properties of WHBS. After eliminating the main nutrients except for starch, the estimated glycemic index (eGI) of the samples rose from 62.56 % to 92.93 %, and the rapidly digested starch content increased from 60.81 % to 98.56 %, respectively. The resistant starch (RS) content, in contrast, dropped from 38.61 % to 0.13 %. Comparatively to lipids, ß-glucan and protein contributed more to the rise in eGI and decline in RS content. The crystalline characteristics of starch were enhanced in the decomposed samples. The samples' gelatinization properties improved, as did the order of the starch molecules. Protein and ß-glucan form a dense matrix on the surface of WHBS particles to inhibit WHBS digestion. In summary, this study revealed the mechanism influencing the digestibility of WHBS from the perspective of endogenous non-starch composition and provided a theoretical basis to develop slow-digesting foods.

A Comprehensive Review of the Effects of Glycemic Carbohydrates on the Neurocognitive Functions Based on Gut Microenvironment Regulation and Glycemic Fluctuation Control.

Improper glycemic carbohydrates (GCs) consumption can be a potential risk factor for metabolic diseases such as obesity and diabetes, which may lead to cognitive impairment. Although several potential mechanisms have been studied, the biological relationship between carbohydrate consumption and neurocognitive impairment is still uncertain. In this review, the main effects and mechanisms of GCs' digestive characteristics on cognitive functions are comprehensively elucidated. Additionally, healthier carbohydrate selection, a reliable research model, and future directions are discussed. Individuals in their early and late lives and patients with metabolic diseases are highly susceptible to dietary-induced cognitive impairment. It is well known that gut function is closely related to dietary patterns. Unhealthy carbohydrate diet-induced gut microenvironment disorders negatively impact cognitive functions through the gut-brain axis. Moreover, severe glycemic fluctuations, due to rapidly digestible carbohydrate consumption or metabolic diseases, can impair neurocognitive functions by disrupting glucose metabolism, dysregulating calcium homeostasis, oxidative stress, inflammatory responses, and accumulating advanced glycation end products. Unstable glycemic status can lead to more severe neurological impairment than persistent hyperglycemia. Slow-digested or resistant carbohydrates might contribute to better neurocognitive functions due to stable glycemic response and healthier gut functions than fully gelatinized starch and nutritive sugars.

Catalytic activity enhancement of 1,4-α-glucan branching enzyme by N-terminal modification.

The 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) has garnered considerable attention for its ability to increase the degree of branching of starch and retard starch digestion, which has great industrial applications. Previous studies have reported that the N-terminal domain plays an important role in the expression and stability of GBEs. To further increase the catalytic ability of Gt-GBE, we constructed five mutants in the N-terminal domain: L19R, L19K, L25R, L25K, and L25A. Specific activities of L25R and L25A were increased by 28.46% and 23.46%, respectively, versus the wild-type Gt-GBE. In addition, the α-1,6-glycosidic linkage ratios of maltodextrin samples treated with L25R and L25A increased to 5.71%, which were significantly increased by 19.96% compared with that of the wild-type Gt-GBE. The results of this study suggest that the N-terminal domain selective modification can improve enzyme catalytic activity, thus further increasing the commercial application of enzymes in food and pharmaceutical industries.

Formation Mechanism of Starch-Based Double Emulsions from the Interfacial Perspective.

Double emulsions are of significant practical value in protecting the core material owing to their multicomponent structure and have thus been applied in various fields, such as food, cosmetics, and drugs. However, the mechanism of double emulsion formation by native starch is not well established. Herein, we demonstrate a facile route to develop type-A, type-B, and type-C double emulsions using native starch and develop an innovative design for a carrier. Interfacial interaction, enthalpy changes of starch, and interfacial properties are key factors governing the formation of double emulsions and controlling the type of double emulsions formed. Therefore, the results of this study provide a better understanding of how and what type of starch-based double emulsions are formed.

Conserved residues at the family and subfamily levels determine enzyme activity and substrate binding in glycoside hydrolase family 13.

Site-directed mutagenesis is a valuable strategy for modifying enzymes, but the lack of understanding of conserved residues regulating glycosidase function hinders enzyme design. We analyzed 1662 enzyme sequences to identify conserved amino acids in maltohexaose-forming amylase at both family and subfamily levels. Several conserved residues at the family level (G37, P45, R52, Y57, D101, V103, H106, G230, R232, D234, E264, H330, D331, and G360) were found, mutations of which resulted in reduced enzyme activity or inactivation. At the subfamily level, several conserved residues (L65, E67, F68, D111, E114, R126, R147, F154, W156, F161, G163, D165, W218H, V342, W345, and F346) were identified, which primarily facilitate substrate binding in the enzyme's active site, as shown by molecular dynamics and kinetic assays. Our findings provide critical insights into conserved residues essential for catalysis and can inform targeted enzyme design in protein engineering.

Bifunctional Mutant of Oligo-1,6-glucosidase for the Production of Glucose from Glucose Mother Liquor.

Glucose mother liquor (GML) is a byproduct of the glucose (G1) crystallization process. However, the presence of maltooligosaccharides and isomaltooligosaccharides within GML imposes limitations on its reutilization. Furthermore, the high concentration of G1 in GML leads to product inhibition of G1-producing enzymes. To overcome these challenges, a variant enzyme called V219A was developed through genetic mutation. The V219A exhibits the ability to hydrolyze both maltooligosaccharides and isomaltooligosaccharides. Product inhibition kinetics showed that the IC50 value of V219A was 7 times higher than that of the wild type. Upon subjecting primary, secondary, and tertiary GML to treatment with V219A, the G1 content exhibited notable increases, reaching 96.88, 95.70, and 90.46%, respectively. These significant findings not only establish an innovative and environmentally conscious approach for G1 production from GML but also provide a promising strategy for enzyme construction that caters to the demands of industrial-scale production.

Impacts of fatty acid type on binding state, fine structure, and in vitro digestion of debranched starch-fatty acid complexes with different debranching degrees.

This study aimed to investigate the effects of fatty acid (FA) type on the binding state, fine structure, and digestibility of debranched maize starch (DMS)-FA complexes with different enzymatic debranching degrees. Maize starch was hydrolyzed by pullulanase for 1 h (DMS1h) and 6 h (DMS6h) and then complexed with seven types of FAs with varying chain lengths and unsaturation degrees, respectively. All the DMS-FA complexes showed V6III-type and B-type crystals. Complex formation greatly increased the relative crystallinity of DMS, but significantly decreased its order degree of short-range structure (p < 0.05). Compared with unsaturated FAs, saturated FAs possessed stronger intermolecular interactions with DMS. DMS6h-FA complexes exhibited a markedly higher complexing degree (p < 0.05) than the corresponding DMS1h-FA complexes. The FA molecules in DMS1h-FA complexes were primarily physically trapped outside the amylose helices, whereas those in DMS6h-FA complexes were mainly weakly bound to the cavity of amylose helices. The resistant starch (RS) content and relative crystallinity of DMS-FA complexes considerably increased with increasing FA chain length. Furthermore, the highest RS content (38.90 %) and relative crystallinity (24.23 %) were observed in DMS6h-FA complexes. The FA unsaturation degree induced little effect on the RS content and long-range structural order of the complexes.

Colon targeted releases and uptakes of paclitaxel loaded in modified porous starch.

Hyaluronic acid can modify porous starch through cross-linking and hydrogen bonding, effectively achieving a paclitaxel entrapment efficiency of ∼92 % and drug loading of ∼23 %. In this study, the pores and intergranular gaps of porous starch were filled with paclitaxel under solvent volatilization, and the enrichment process and its characteristics were recorded using a microscope. The paclitaxel-loaded particles were coated with chitosan-phytic acid to target the colon. In vivo imaging in mice showed that the capsule released paclitaxel in the colon rather than in the upper digestive tract, and the paclitaxel distribution in the main organs at 24 h was significantly lower than that of raw paclitaxel. Hyaluronic acid-modified porous starch can target cancer cells. Cell internalization of paclitaxel mediated by hyaluronic acid was approximately 1.97 times that of raw paclitaxel, higher than that of receptor-shielded cells and cells incubated with unmodified carriers, as evidenced by the accumulation of fluorescent paclitaxel in the nucleus and marked cell apoptosis. The hyaluronic acid-modified porous starch system is an effective method for the high-load and targeted release of hydrophobic anticancer drugs.