Catalytic Mode and Product Specificity of an α-Agarase Reveal Its Direct Catalysis for the Production of Agarooligosaccharides.

Many α-agarases have been characterized and are utilized for producing agarooligosaccharides through the degradation of agar and agarose, which are considered valuable for applications in the food and medicine industries. However, the catalytic mechanism and product transformation process of α-agarase remain unclear, limiting further enzyme engineering for industrial applications. In this study, an α-agarase from Catenovulum maritimus STB14 (Cm-AGA) was employed to degrade agarose oligosaccharides (AGOs) with varying degrees of polymerization (DPs) to investigate the catalytic mechanism of α-agarases. The results demonstrated that Cm-AGA could degrade agarose into agarotetraose and agarohexaose. The reducing ends of agarotetraose and agarohexaose spontaneously release unstable 3,6-anhydro-α-l-galactose molecules, which were further degraded into agarotriose and agaropentose. Cm-AGA cannot act on α-1,3-glucoside bonds in agarotriose, agarotetraose, neoagarobiose, and neoagarotetraose but can act on AGOs with a DP greater than four. The product analysis was further verified by ß-galactosidase hydrolysis, which specifically cleaves the non-reducing glycosidic bond of agarooligosaccharides. Multiple sequence alignment results showed that two conserved residues, Asp994 and Glu1129, were proposed as catalytic residues and were further identified by site-directed mutagenesis. Molecular docking of Cm-AGA with agaroheptose revealed the potential substrate binding mode of the α-agarase. These findings enhance the understanding of Cm-AGA's catalytic mode and could guide enzyme engineering for modulating the production of agarooligosaccharides.

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.

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.

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.

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.

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.

Designing a Specific Pretreatment on Corn Starch to Facilitate Enzymatic Rearrangement of Glycosidic Bonds for Efficiently Reducing Starch Digestibility.

Bacterial 1,4-α-glucan branching enzymes (GBEs) provide a viable strategy for glycosidic bond rearrangement in starch and regulation of its digestion rate. However, the exponential increase in paste viscosity during starch gelatinization has a detrimental effect on the catalytic action of GBEs, thereby limiting productivity and product performance. Here, we designed an enzymatic treatment on corn starch granules by the GBE from Rhodothermus obamensis STB05 (Ro-GBE) prior to the glycosidic bond rearrangement of gelatinized starch catalyzed using the GBE from Geobacillus thermoglucosidans STB02 (Gt-GBE). Specifically, a moderate amount of Ro-GBE was required for the pretreatment stage. The dual GBE modification process enabled the treatment of more concentrated starch slurry (up to 20%, w/w) and effectively reduced starch digestibility. The resulting product contained a rapidly digestible starch fraction of 66.0%, which was 11.4% lower than that observed in the single Gt-GBE-modified product. The mechanistic investigation showed that the Ro-GBE treatment promoted swelling and gelatinization of starch granules, reduced starch paste viscosity, and increased the mobility of water molecules in the starch paste. It also created a preferable substrate for Gt-GBE. These changes improved the transglycosylation efficiency of Gt-GBE. These findings provide useful guidance for designing an efficient process to regulate starch digestibility.

Distribution of Aromatic Amino Acid Residues in Substrate-Binding Regions Modulates Substrate Specificity of Microbial Debranching Enzymes.

Debranching enzymes (DBEs) directly hydrolyze α-1,6-glucosidic linkages in glycogen, starch, and related polysaccharides, making them important in the starch processing industry. However, the ambiguous substrate specificity usually restricts synergistic catalysis with other amylases for improving starch utilization. Herein, a glycogen-debranching enzyme from Saccharolobus solfataricus (SsGDE) and two isoamylases from Pseudomonas amyloderamosa (PaISO) and Chlamydomonas reinhardtii (CrISO) were used to investigate the molecular mechanism of substrate specificity. Along with the structure-based computational analysis, the aromatic residues in the substrate-binding region of DBEs played an important role in binding substrates. The aromatic residues in SsGDE appeared clustered, contributing to a small substrate-binding region. In contrast, the aromatic residues in isoamylase were distributed dispersedly, forming a large active site. The distinct characteristics of substrate-binding regions in SsGDE and isoamylase might explain their substrate preferences for maltodextrin and amylopectin, respectively. By modulating the substrate-binding region of SsGDE, variants Y323F and V375F were obtained with significantly enhanced activities, and the activities of Y323F and V375F increased by 30 and 60% for amylopectin, and 20 and 23% for DE4 maltodextrin, respectively. This study revealed the molecular mechanisms underlying the substrate specificity for SsGDE and isoamylases, providing a route for engineering enzymes to achieve higher catalytic performance.

Physicochemical characterizations, α-amylase inhibitory activities and inhibitory mechanisms of five bacterial exopolysaccharides.

Inhibiting pancreatic α-amylase activity can decrease the release rate of glucose, thereby delaying postprandial blood glucose. This study aimed to investigate the physicochemical properties and porcine pancreatic α-amylase (PPA) inhibitory activities of five bacterial exopolysaccharides (EPSs). We also aimed to analyze the differences of their inhibitory activities, exploring the inhibition mechanism between EPSs and PPA. Five EPSs had a low molecular weight (55-66 kDa), which were mainly composed of mannose and glucose with total content exceeding 86 %. The IC50 values of five EPSs (0.162-0.431 mg/mL) were significantly lower than that of acarbose (0.763 mg/mL), indicating that the inhibitory effects of five EPSs on PPA were stronger than acarbose, especially the EPS from Bacillus subtilis STB22 (BS-EPS). Moreover, BS-EPS was a mixed-type inhibitor, whereas other EPSs were noncompetitive inhibitors of PPA. Five EPSs quenched the fluorophore of PPA by the mixed quenching or apparent static quenching. Interestingly, BS-EPS showed stronger binding affinity to PPA than other EPSs. It can be speculated that EPSs with low molecular weight, high carboxylic acid content, and α-glycosidic bond exhibited high PPA inhibitory activity. These results suggest that BS-EPS can effectively inhibit PPA activity and has potential applications in reducing postprandial hyperglycemia.

Conserved residues Glu and Phe at substrate binding groove of α-1,6-glucanases modulate branch of the product.

Understanding what amino acids in α-1,6-glucanases target α-1,6 glycosidic bonds of polysaccharides is timely and important for generating products with branch structure. With this objective, we investigated 330 sequences from seven subfamilies to excavate amino acids for recognition or catalysis of α-1,6 glycosidic bonds. Computational analysis identified two amino acids, E343 and W521, trigger α-1,6 glycosidic bond specificity of enzymes. To explore the effect of E343 and W521 on the product structure, several engineered mutants were studied in our research. Product structural analysis showed that the ratio of amylose and amylopectin is obviously different. The catalytic mechanism revealed that the bulky aromatic side chain is a trigger that controls the ratio of branch glucans. The E148 acts as a proton donor to regulate the generation of branched structures in the product during transglycosidation of the glucan branching enzyme (GBE).

Designing liquefaction and saccharification processes of highly concentrated starch slurry: Challenges and recent advances.

Starch-based sugars are an important group of starch derivatives used in food, medicine, chemistry, and other fields. The production of starch sugars involves starch liquefaction and saccharification processes. The production cost of starch sugars can be reduced by increasing the initial concentration of starch slurry. However, the usage of the highly concentrated starch slurry is characterized by challenges such as low reaction efficiency and poor product performance during the liquefaction and saccharification processes. In this study, we endeavored to provide a reference guide for improving high-concentration starch sugar production. Thus, we reviewed the effects of substrate concentration on the starch sugar production process and summarized several potential strategies. These regulation strategies, such as physical field pretreatment, complex enzyme-assisted, and temperature control, can significantly increase the starch concentration and mitigate the challenges of using highly concentrated starch slurry. We believe that highly concentrated starch sugar production will achieve a qualitative leap in the future. This review provides theoretical guidance and highlights the importance of high concentration in starch-based sugar production. Further studies are needed to explore the fine structure and enzyme attack mode during the liquefaction and saccharification processes to regulate the production of more targeted products.

Short-Clustered Maltodextrin Activates Ileal Glucose-Sensing and Induces Glucagon-like Peptide 1 Secretion to Ameliorate Glucose Homeostasis in Type 2 Diabetic Mice.

Reconstructing molecular structure is an effective approach to attenuating glycemic response to starch. Previously, we rearranged α-1,4 and α-1,6-glycosidic bonds in starch molecules to produce short-clustered maltodextrin (SCMD). The present study revealed that SCMD slowly released glucose until the distal ileum. The activated ileal glucose-sensing enabled SCMD to be a potent inducer for glucagon-like peptide-1 (GLP-1). Furthermore, SCMD was found feasible to serve as the dominant dietary carbohydrate to rescue mice from diabetes. Interestingly, a mixture of normal maltodextrin and resistant dextrin (MD+RD), although it caused an attenuated glycemic response similar to that of SCMD, failed to ameliorate glucose homeostasis because it hardly induced GLP-1 secretion. The serum GLP-1 levels seen in MD+RD-fed mice (5.25 ± 1.51 pmol/L) were significantly lower than those seen in SCMD-fed mice (8.25 ± 2.01 pmol/L, p < 0.05). Further investigation revealed that the beneficial effects of SCMD could be abolished by a GLP-1 receptor (GLP-1R) antagonist. These results identify GLP-1R signaling as a critical contributor to SCMD-exerted health benefits and highlight the role of ileal glucose-sensing in designing dietary carbohydrates.

Rice noodle quality is structurally driven by the synergistic effect between amylose chain length and amylopectin unit-chain ratio.

Chewiness, slipperiness, and rapid rehydration are satisfactory qualities for rice noodles. These qualities are dependent on rice starch structure. However, the synergistic effect of fine structure of amylose (AM) and amylopectin (AP) on rice noodle quality remains unclear. In this work, six rice varieties, three from early indica and three from late indica, with similar amylose content for each pair were analyzed to assess the synergistic effects between AM and AP fine structures. The results showed that the combination of amylose long-chains and a low amylopectin unit-chain (APC) ratio favored the maintenance of relatively intact starch granules, resulting in a higher die expansion ratio and improvement of the mechanical properties of rice noodles (Hardness varied from 403 g to 1627 g). Meanwhile, higher die expansion was associated with a larger rehydration ratio. These results suggest that the synergistic effect between AM and AP fine structures significantly affects rice noodles' quality.

Highly branched starch accelerates the restoration of edible quality of dried rice noodles during rehydration.

Rice noodle with excellent edible quality usually needs a dense gel network structure, but the dense structure is detrimental to the entry of water molecules during dried rice noodle rehydration. To combine the conflict requirements, we described the branching modification of rice starch using 1,4- α- glucan branching enzyme (GBE). Highly branched starch with a short cluster structure reduced fractal dimension and crystallite thickness while increasing mesh size and hydrophilicity of rice noodle gel network. Rice noodles derived from rice soaked with 128 U/g of GBE had a desirable rehydration time (370 s), which was reduced by 39.84% compared to the control. Meanwhile, the shorter double helix formed by the short cluster contributed to the improved short-range order, resulting in GBE-modified rice noodles with significantly higher tensile strength than control. These findings demonstrate that manipulating the branching degree of starch is an effective method for producing high-quality instant rice noodles.

Perspectives on evaluating health effects of starch: Beyond postprandial glycemic response.

Starch is an important dietary carbohydrate in the human diet and is greatly associated with human health. The health effects of starch are classically evaluated by postprandial glycemic response. However, glycemic response is the test result of blood glucose level and sometimes fails to perfectly describe the health effects exerted by starch. Therefore, other factors, besides glycemic response, merit consideration. Herein, we endeavor to provide some insights into the description of health effects exerted by starch. For this purpose, we summarize advances in recent studies to support the crucial roles of glucose kinetics, insulin response, and gut hormones release. A moderate postprandial insulin response and an enhanced release of several specific gut hormones are critical characteristics of a healthier starch, such as those slowly digested till the distal ileum. It is also hoped that further studies can develop feasible methods to produce tailor-made starches with individualized health effects.

Beneficial Effects of Three Dietary Cyclodextrins on Preventing Fat Accumulation and Remodeling Gut Microbiota in Mice Fed a High-Fat Diet.

Globally, obesity and its metabolic complications, which are intimately linked to diet, are major public health problems. Cyclodextrins (CDs) are cyclic oligosaccharides consisting of (α-1,4)-linked D-glucopyranose units that can reduce fat bioavailability and affect metabolism by improving intestinal flora as prebiotics. We compared the effects of three CDs on preventing fat accumulation and remodeling gut microbiota in a high-fat diet-fed C57BL/6J mouse model. α-CD maximized energy expenditure by 12.53%, caused the RER value to drop from 0.814 to 0.788, and increased the proportion of fatty acid oxidation for energy supply. ß-CD supplementation resulted in a marked 24.53% reduction in weight gain and a decrease in epididymal-fat-relative weight from 3.76% to 2.09%. It also minimized ectopic fat deposition and improved blood lipid parameters. γ-CD maximized the concentration of SCFAs in the cecum from 6.29 to 15.31 µmol/g. All three CDs reduced the abundance ratio of Firmicutes and Bacteroidetes to a low-fat diet level, increased the abundance of Lactobacillus and Akkermansia, and reduced the abundance of Allobaculum and Ruminococcus. These findings imply that a combination of multiple CDs may exert superior effects as a potential strategy for obesity prevention.

Two 1,4-α-glucan branching enzymes successively rearrange glycosidic bonds: A novel synergistic approach for reducing starch digestibility.

Enzymatically rearranging α-1,4 and α-1,6 glycosidic bonds in starch is a green approach to regulating its digestibility. A two-step modification process successively catalyzed by 1,4-α-glucan branching enzymes (GBEs) from Rhodothermus obamensi STB05 (Ro-GBE) and Geobacillus thermoglucosidans STB02 (Gt-GBE) was investigated as a strategy to reduce the digestibility of corn starch. This dual GBE modification process caused a reduction of 25.8 % in rapidly digestible starch fraction in corn starch, which were more effective than single GBE-catalyzed modification with the same duration. Structural analysis indicated that the dual GBE modified product contained higher branching density, more abundant short branches, and shorter external chains than those in single GBE-modified product. These results demonstrated that a moderate Ro-GBE treatment prior to starch gelatinization caused several suitable alterations in starch molecules, which promoted the transglycosylation efficiency of the following Gt-GBE treatment. This dual GBE-catalyzed modification process offered an efficient strategy for regulating starch digestibility.

An Innovative Short-Clustered Maltodextrin as Starch Substitute for Ameliorating Postprandial Glucose Homeostasis.

Dietary starch is usually associated with elevated postprandial glycemic response. This is a potential risk factor of type 2 diabetes. Here, a 1,4-α-glucan branching enzyme (GBE) was employed to reassemble α-1,4 and α-1,6 glycosidic bonds in starch molecules. Structural characterization showed that GBE-catalyzed molecular reassembly created an innovative short-clustered maltodextrin (SCMD), which showed a dense internal framework along with shortened external chains. Such short-clustered molecules obstructed digestive enzymes attack and displayed dramatically reduced digestibility. Therefore, SCMD was served as a dietary starch substitute to improve postprandial glucose homeostasis. A 22.3% decrease in glycemic peak was therefore detected in ICR mice following SCMD intake (10.7 mmol/L), compared with that in the control (13.8 mmol/L). Moreover, an attenuated insulin response (40.5% lower than that in control) to SCMD intake was regarded suitable for diabetes management. These novel discoveries demonstrate that enzymatically rebuilding starch molecules may be a meaningful strategy for diabetes management.