N1019D Mutant of Limosilactobacillus reuteri 121 4,6-α-Glucanotransferase GtfB Significantly Improved Catalytic Activity.

Limosilactobacillus reuteri 121 4,6-α-glucanotransferase GtfB (Lr 121 GtfB), belonging to glycoside hydrolase family 70 (GH70), synthesizes linear isomalto/malto polysaccharides having (α1→6) linkages attached to the nonreducing ends of (α1→4) linked maltose oligosaccharide segments using starch or maltodextrin as a substrate. Since Lr 121 GtfB has low catalytic activity and efficiency, it leads to substrate regeneration and reduced substrate utilization. In this study, we superimposed the crystal structure of Lr 121 GtfB-ΔNΔV with that of L. reuteri NCC 2613 GtfB-ΔNΔV (Lr 2613 GtfB-ΔNΔV) to identify the acceptor binding subsites +1 to +3 and constructed five single-residue mutants and a random mutagenesis of N1019. Compared with the wild-type, N1019D Lr 121 GtfB-ΔN did not alter the product specificity, increased the catalytic activity and efficiency by 420 and 590%, respectively, and maintained >80% relative activity in the pH 3.5-6.5 interval. The findings will contribute to the industrial application of Lr 121 GtfB and provide new solutions for starch synthesis of higher value derivatives.

Enhancing the anti-thixotropic properties of waxy maize starch modified by different α-amylases and its underlying molecular mechanism.

The large thixotropy of the starch-thickened foods is often unfavorable in many applications. This study examined the contribution of the proportion of amylopectin chain length to time-dependence of starch gels. The α-amylase (AM) from Bacillus stearothermophilus and maltogenic α-amylase (MA) from Bacillus subtilis were used to trim amylopectin in different reaction patterns. HPLC, HPAEC and IBC data suggested AM attacked B-chains (DP 12-36), causing an increment in number of the chains with DP 6-12, whereas MA primarily trimmed the short B-chains (DP 12-18) and partial A-chains (DP 9-12) to generate short chains with DP 6-9. Interestingly, the recovery of AM-gels was faster than MA-gels at the same degree of hydrolysis when subjected to shear according to the linear correlation analysis. When releasing the same mass of sugar, shortening of the long internal chains played an important role in reducing time dependence of starch gel rather than the external side chains. Possible models were proposed to illustrate the differences in the mechanism of rapid-recovery caused by different side-chain distributions. The outcome provided a new perspective to regulate the thixotropy behavior of starch through enzyme strategies in the granular state.

Insights into the Structure-Function Relationship of GH70 GtfB α-Glucanotransferases from the Crystal Structure and Molecular Dynamic Simulation of a Newly Characterized Limosilactobacillus reuteri N1 GtfB Enzyme.

α-Glucanotransferases of the CAZy family GH70 convert starch-derived donors to industrially important α-glucans. Here, we describe characteristics of a novel GtfB-type 4,6-α-glucanotransferase of high enzyme activity (60.8 U mg-1) from Limosilactobacillus reuteri N1 (LrN1 GtfB), which produces surprisingly large quantities of soluble protein in heterologous expression (173 mg pure protein per L of culture) and synthesizes the reuteran-like α-glucan with (α1 → 6) linkages in linear chains and branch points. Protein structural analysis of LrN1 GtfB revealed the potential crucial residues at subsites -2∼+2, particularly H265, Y214, and R302, in the active center as well as previously unidentified surface binding sites. Furthermore, molecular dynamic simulations have provided unprecedented insights into linkage specificity hallmarks of the enzyme. Therefore, LrN1 GtfB represents a potent enzymatic tool for starch conversion, and this study promotes our knowledge on the structure-function relationship of GH70 GtfB α-glucanotransferases, which might facilitate the production of tailored α-glucans by enzyme engineering in future.

Progress in cyclodextrins as important molecules regulating catalytic processes of glycoside hydrolases.

Cyclodextrins (CDs) are important starch derivatives and commonly comprise α-, ß-, and γ-CDs. Their hydrophilic surface and hydrophobic inner cavity enable regulation of enzyme catalysis through direct or indirect interactions. Clarifying interactions between CDs and enzyme is of great value for enzyme screening, mechanism exploration, regulation of catalysis, and applications. We summarize the interactions between CDs and glycoside hydrolases (GHs) according to two aspects: 1) CD as products, substrates, inhibitors and activators of enzymes, directly affecting the reaction process; 2) CDs indirectly affecting the enzymatic reaction by solubilizing substrates, relieving substrate/product inhibition, increasing recombinant enzyme production and storage stability, isolating and purifying enzymes, and serving as ligands in crystal structure to identify functional amino acid residues. Additionally, CD enzyme mimetics are developed and used as catalysts in traditional artificial enzymes as well as nanozymes, making the application of CDs no longer limited to GHs. This review concerns the regulation of GHs catalysis by CDs, and gives insights into research on interactions between enzymes and ligands.

Identification of a novel starch-converting GtfB enzyme from the Fructilactobacillus sanfranciscensis TMW11304 to reduce the viscoelasticity and retrogradation of tapioca starch.

Starch-converting α-glucanotransferases are efficient enzymatic toolkits for the biosynthesis of diverse α-glucans, which hold vast application potential in the food industry. In this work, we identified a novel GtfB protein from Fructilactobacillus sanfranciscensis TMW11304 (FsTMW11304 GtfB) in NCBI. Although this enzyme was highly conserved in motifs I-IV with those isomalto-maltopolysaccharides (IMMPs)-producing GtfB α-glucanotransferases, it possessed distinct deletions and mutations in two crucial loops shaping the active site. Hence, unlike those GtfB enzymes, FsTMW11304 GtfB not only exhibited excellent 4,6-α-glucanotransferase activity on amylose to generate atypically low-molecular-weight IMMPs with consecutive linear (α1 â†’ 6) linkages up to 48 %, but also held good capability towards branched substrates. Besides, compared with the control, the treatment by FsTMW11304 GtfB reduced the storage/loss modulus of granular and gelatinized tapioca starches (TS) by 12.0 %/17.9 % and 91.4 %/82.9 %, respectively, indicating that the rigidity of the gel structure was attenuated to different degrees in the two reaction systems. Furthermore, the setback viscosity observed in the gelatinized TS modified by FsTMW11304 GtfB was only 5 % of that observed in the control group, suggesting the short-term anti-retrogradation property has been substantially improved. Thus, FsTMW11304 GtfB represents a meaningful addition to the α-glucanotransferases in GH70 family, which expands the repertoire of diverse α-glucans synthesized from starch and facilitates the understanding of the structure-function relationship of the GtfB α-glucanotransferases.

Exploring a GtfB-Type 4,6-α-Glucanotransferase to Synthesize the (α1 → 6) Linkages in Linear Chain and Branching Points from Amylose and Enhance the Functional Property of Granular Corn Starches.

Starch-converting α-glucanotransferases of glycoside hydrolase family 70 (GH70) are promising enzymatic tools for the production of diverse α-glucans with (potential) commercial applications in food and health and as biomaterials. In this study, a novel GtfB enzyme from Weissella confusa MBF8-1 was screened in the National Center for Biotechnology Information (NCBI) nonredundant protein database. The enzyme (named WcMBF8-1 GtfB) displayed high conservation in motifs I-IV with other GtfB enzymes but possessed unique variations in several substrate-binding residues. Structural characterizations of its α-glucan products revealed that WcMBF8-1 GtfB exhibited an atypical 4,6-α-glucanotransferase activity and was capable of catalyzing, by cleaving off (α1 → 4)-linkages in starch-like substrates and the synthesis of linear (α1 → 6) linkages and (α1 → 4,6) branching points. The product specificity enlarges the diversity of α-glucans and facilitates recognition of the determinants of the linkage specificity in GtfB enzymes. Furthermore, the contents of slowly digestible starch and resistant starch of granular corn starches, modified by WcMBF8-1 GtfB, increased by 6.7%, which suggested the potential value for the utilization of WcMBF8-1 GtfB to prepare "clean-label" starch ingredients with improved functional attributes.

Analysis of the action pattern of sequential α-amylases from B. stearothermophilus and B. amyloliquefaciens on highly concentrated soluble starch.

Hydrolysis of highly concentrated soluble starch (60%, w/w) was performed using sequential α-amylases from Bacillus stearothermophilus (T, 0.2%, w/w) and Bacillus amyloliquefaciens (B, 0.1%, w/w) to identify their possible action patterns. We found that T reduced the average molecular weight (Mw) of soluble starch from 52,827 Da to 31,914 Da and significantly affected its branched chain length. Compared with soluble starch, the chains with DP 6-12 and DP ≥ 13 in the T samples were diminished by 46% and 96%, respectively. This resulted in an attenuation in the proportions of exterior and inner chains, as well as low iodine binding capacity of the hydrolysates. In contrast, a slower decrease in the average Mw of soluble starch occurred after TB incubation, and the level of DP 6-12 further lowered, causing a gradual decline in the iodine binding capacity of the hydrolysates. Gathered data revealed an unusual action pattern of sequential α-amylase treatment at high substrate concentrations. Bacillus stearothermophilus α-amylase exhibited more pronounced endo-hydrolysis of amylopectin, whereas the attack of Bacillus amyloliquefaciens α-amylase on the exterior chains was enhanced in amylopectin residues. These findings suggest that the synergy of various α-amylases is an effective strategy to promote the dextrinization of highly concentrated starch and finely modify the molecular structure of starch.

Effects of maltogenic α-amylase on physicochemical properties and edible quality of rice cake.

Maltogenic α-amylase (MA) are commercially used in the baking industry to retard starch retrogradation. However, whether MA can be used to modify rice flour during the fermentation process to improve the quality of rice flour remains unclear. In this study, MA was introduced during rice cake (RC) processing, and the modification effect and underlying mechanism were explored. Mn showed a decreasing trend except for 4.0 × 10-3 U/g sample. Chain length distribution data showed that MA effectively hydrolyzed long chains in amylopectin and increased the concentration of amylopectin chain length with a degree of polymerization of ≤ 9. High-performance liquid chromatography results suggested that the maltose content increased to 3.14% at an MA concentration of 9.5 × 10-3 U/g, which affected the fermentation effect of MA-treated RC. MA effectively reduced the viscosity of RC, and the gelatinization enthalpy of RC changed to 0.835 mJ/mg. MA also reduced the hardness and chewiness of RC after storage for 7 d. Moreover, rapidly digestible starch and slowly digestible starch contents of MA-treated RC decreased and increased, respectively, and resistant starch contents were remained unchanged. These results indicate that MA exerts a significant and effective antiretrogradation effect on RC. Combining the above results with sensory evaluation findings, an MA concentration of 4.0 × 10-3 U/g was the best supplemental concentration for obtaining RC with better edible quality. These findings suggest that MA treatment to rice flour during the fermentation process not only preserved the edible quality of RC but also retarded its retrogradation, thus, providing a novel processing method for the industrial production of RC.

Adipocyte-Derived Exosomal NOX4-Mediated Oxidative Damage Induces Premature Placental Senescence in Obese Pregnancy.

Background: A recent study has reported that maternal obesity is linked to placental oxidative damage and premature senescence. NADPH oxidase 4 (NOX4) is massively expressed in adipose tissue, and its induced reactive oxygen species have been found to contribute to cellular senescence. While, whether, in obese pregnancy, adipose tissue-derived NOX4 is the considerable cause of placental senescence remained elusive. Methods: This study collected term placentas from obese and normal pregnancies and obese pregnant mouse model was constructed by a high fat diet to explore placental senescence. Furthermore, adipocyte-derived exosomes were isolated from primary adipocyte medium of obese and normal pregnancies to examine their effect on placenta functions in vivo and vitro. Results: The placenta from the obese group showed a significant increase in placental oxidative damage and senescence. Exosomes from obese adipocytes contained copies of NOX4, and when cocultured with HTR8/SVneo cells, they induced severe oxidative damage, cellular senescence, and suppressed proliferation and invasion functions when compared with the control group. In vivo, adipocyte-derived NOX4-containing exosomes could induce placental oxidative damage and senescence, ultimately leading to adverse pregnancy outcomes. Conclusion: In obesity, adipose tissue can secrete exosomes containing NOX4 which can be delivered to trophoblast resulting in severe DNA oxidative damage and premature placental senescence, ultimately leading to adverse pregnancy outcomes.

Regulation of multi-scale structures and retrogradation property of A- and B-type wheat starch granules with maltogenic α-amylase.

Maltogenic α-amylase (MA) is widely used to modify starch for improving properties. In this work, A- and B-type starches were separated from wheat completely and submitted to MA hydrolysis. Firstly, research in morphological features (SEM) suggested that MA treatment did not destroy the granule integrity. Next, crystalline features from XRD and SAXS assay showed that MA treatment did not change the crystal form, but deceased crystalline sheet (dc) and amorphous sheet (da) thickness in both modified starches. And amorphous sheet was more severely destroyed at higher MA dosage than crystalline sheet. Then changes in molecule structure (HPAEC) showed that MA mainly acted on sides chains with degree of polymerization 12-24 in amylopectin for both starches, resulting in a reduce in peak, trough, and final viscosity obtained from RVA assay. At last, RVA and DSC assay showed that the short-term and long-term retrogradation were retarded in both modified starches. This range of techniques covered changes in multi-scale structures and retrogradation property resulted from MA treatment on both starches, which provided references for studying the changes in structures and properties of MA modified starch granules and provided an important method for retarding retrogradation of starchy foods without gelatinization processing.

miR-101-5p suppresses trophoblast cell migration and invasion via modulating the DUSP6-ERK1/2 axis in preeclampsia.

PURPOSE: Dysregulated behaviors of trophoblast cells leading to defective placentation are considered the main cause of preeclampsia (PE). Abnormal miRNA expression profiles have been observed in PE placental tissue, indicating the significant role of miRNAs in PE development. This study aimed to investigate the expression of miR-101-5p in PE placental tissue and its biological functions. METHODS: The expression of miR-101-5p in placental tissue was detected by quantitative real-time PCR (qRT-PCR). The localization of miR-101-5p in term placental tissue and decidual tissue was determined by the fluorescence in situ hybridization (FISH)-immunofluorescence (IF) double labeling assay. The effect of miR-101-5p on the migration, invasion, proliferation, and apoptosis of the HTR8/SVneo trophoblast cells was investigated. Online databases combined with transcriptomics were used to identify potential target genes and related pathways of miR-101-5p. Finally, the interaction between miR-101-5p and the target gene was verified by qRT-PCT, WB, dual-luciferase reporter assay, and rescue experiments. RESULTS: The study found that miR-101-5p was upregulated in PE placental tissue compared to normal controls and was mainly located in various trophoblast cell subtypes in placental and decidual tissues. Overexpression of miR-101-5p impaired the migration and invasion of HTR8/SVneo cells. DUSP6 was identified as a potential downstream target of miR-101-5p. The expression of miR-101-5p was negatively correlated with DUSP6 expression in HTR8/SVneo cells, and miR-101-5p directly bound to the 3' UTR region of DUSP6. DUSP6 upregulation rescued the migratory and invasive abilities of HTR8/SVneo cells in the presence of miR-101-5p overexpression. Additionally, miR-101-5p downregulated DUSP6, resulting in enhanced ERK1/2 phosphorylation. CONCLUSION: This study revealed that miR-101-5p inhibits the migration and invasion of HTR8/SVneo cells by regulating the DUSP6-ERK1/2 axis, providing a new molecular mechanism for the pathogenesis of PE.

Exploring the roles of amylopectin in starch modification with Limosilactobacillus reuteri 121 4,6-α-glucanotransferase via developed methods.

Limosilactobacillus reuteri 121 4,6-α-glucanotransferase (GtfBΔN) modifies starch by cleaving (α1 â†’ 4) linkages and introducing non-branched (α1 â†’ 6) linkages to produce functional starch derivatives. Research has mainly focused on GtfBΔN converting amylose (linear substrate), whereas the conversion of amylopectin (branched substrate) has not been studied in detail. In this study, we used GtfBΔN to understand amylopectin modification and performed a set of experiments to analyze this modification pattern. The donor substrates were segments from the non-reducing ends to the nearest branch point in amylopectin as shown from the results of the chain length distribution of GtfBΔN-modified starches. Decreased and increased contents of ß-limit dextrin and reducing sugars, respectively, during the incubation of ß-limit dextrin with GtfBΔN indicated that the segments from the reducing end to the nearest branch point in amylopectin act as donor substrates. Dextranase was involved in the hydrolysis of the GtfBΔN conversion products of three different substrates groups, maltohexaose (G6), amylopectin, and G6 plus amylopectin. No reducing sugars were detected, therefore, amylopectin was not used as an acceptor substrate, and no non-branched (α1 â†’ 6) linkages were introduced into it. Thus, these methods provide a reasonable and effective approach to studying GtfB-like 4,6-α-glucanotransferase in analyzing the roles and contribution of branched substrates.

Designing starch derivatives with desired structures and functional properties via rearrangements of glycosidic linkages by starch-active transglycosylases.

Modification of starch by transglycosylases from glycoside hydrolase families has attracted much attention recently; these enzymes can produce starch derivatives with novel properties, i.e. processability and functionality, employing highly efficient and safe methods. Starch-active transglycosylases cleave starches and transfer linear fragments to acceptors introducing α-1,4 and/or linear/branched α-1,6 glucosidic linkages, resulting in starch derivatives with excellent properties such as complexing and resistance to digestion characteristics, and also may be endowed with new properties such as thermo-reversible gel formation. This review summarizes the effects of variations in glycosidic linkage composition on structure and properties of modified starches. Starch-active transglycosylases are classified into 4 groups that form compounds: (1) in cyclic with α-1,4 glucosidic linkages, (2) with linear chains of α-1,4 glucosidic linkages, (3) with branched α-1,6 glucosidic linkages, and (4) with linear chains of α-1,6 glucosidic linkages. We discuss potential processability and functionality of starch derivatives with different linkage combinations and structures. The changes in properties caused by rearrangements of glycosidic linkages provide guidance for design of starch derivatives with desired structures and properties, which promotes the development of new starch products and starch processing for the food industry.

Increasing Protein Content of Rice Flour with Maintained Processability by Using Granular Starch Hydrolyzing Enzyme.

Rice flour (RF) has become a promising food material. In the present study, RF with higher protein content was prepared using a granular starch hydrolyzing enzyme (GSHE). Particle size, morphology, crystallinity, and molecular structures of RF and rice starch (RS) were characterized to establish a hydrolytic mechanism; thermal, pasting, and rheological properties were determined to evaluate processability using differential scanning calorimetry (DSC), rapid viscosity analysis (RVA), and rheometer, respectively. The GSHE treatment resulted in pinholes, pits, and surface erosion through sequential hydrolysis of crystalline and amorphous areas on the starch granule surface. The amylose content decreased with hydrolysis time, while the very short chains (DP < 6) increased rapidly at 3 h but decreased slightly later. After hydrolysis for 24 h, the protein content in RF increased from 8.52% to 13.17%. However, the processability of RF was properly maintained. Specifically, the data from DSC showed that the conclusion temperature and endothermic enthalpy of RS barely changed. The result of rapid RVA and rheological measurement indicated that RF paste viscosity and viscoelastic properties dropped rapidly after 1 h hydrolysis and thereafter recovered slightly. This study provided a new RF raw material useful for improving and developing RF-based foods.

Deciphering the structural and functional characteristics of an innovative small cluster branched α-glucan produced by sequential enzymatic synthesis.

Highly branched α-glucan (HBAG) proved to be a promising material as an osmotic agent in peritoneal dialysis solutions. However, high resistance of HBAG to amylolytic enzymes might be a potential drawback for peritoneal dialysis due to its high degree of branching (20-30 %). To address this issue, we designed a small-clustered α-glucan (SCAG) with a relatively low molecular weight (Mw) and limited branching. Structural characteristics revealed that SCAG was successfully synthesized by modifying waxy rice starch (WRS) using sequential maltogenic α-amylase (MA) and starch branching enzyme (BE). The Mw of SCAG was 1.40 × 105 Da, and its (α1 â†’ 6) bonds ratio was 8.93 %, which was below that of HBAG. A relatively short branch distribution was observed in SCAG (CL = 6.27). Short-range orderliness of WRS was reduced from 0.749 to 0.322 with the MABE incubation. Additionally, SCAG had an extremely low viscosity (~12 cP) and nearly no retrogradation. Although the resistance of SCAG to amylolytic enzymes was enhanced by 15.22 % compared with native WRS, the extent was significantly lower than that of HBAG in previous studies. These new findings demonstrate the potential of SCAG as a novel functional α-glucan in food and pharmaceutical applications.

Cost-effective and controllable synthesis of isomalto/malto-polysaccharides from ß-cyclodextrin by combined action of cyclodextrinase and 4,6-α-glucanotransferase GtfB.

Isomalto/malto-polysaccharides (IMMPs) derived from malto-oligosaccharides such as maltoheptaose (G7) are elongated non-branched gluco-oligosaccharides produced by 4,6-α-glucanotransferase (GtfB). However, G7 is expensive and cumbersome to produce commercially. In this study, a cost-effective enzymatic process for IMMPs synthesis is developed that utilizes the combined action of cyclodextrinase from Palaeococcus pacificus (PpCD) and GtfB-ΔN from Limosilactobacillus reuteri 121 to convert ß-cyclodextrin into IMMPs with a maximum yield (16.19 %, w/w). The purified IMMPs synthesized by simultaneous or sequential treatments, designated as IMMP-Sim and IMMP-Seq, possess relatively high contents of α-(1 â†’ 6) glucosidic linkages. By controlling the release of G7 and smaller malto-oligosaccharides by PpCD, IMMP-Seq was obtained of DP varying from 12.9 to 29.5. Enzymatic fingerprinting revealed different linkage-type distribution of α-(1 â†’ 6) linked segments with α-(1 â†’ 4) segments embedded at the reducing end and middle part. The proportion of α-(1 â†’ 6) segments containing the non-reducing end was 56.76 % for IMMP-Sim but 28.98 % for IMMP-Seq. Addition of G3 or G4 as specific acceptors resulted in IMMPs exhibiting low polydispersity. This procedure can be applied as a novel bioprocess that does not require costy high-purity malto-oligosaccharides and with control of the average DP of IMMPs by adjusting the substrate composition.

A practical approach to producing the single-arm linear dextrin, a chimeric glucosaccharide containing an (α-1 → 4) linked portion at the nonreducing end of an (α-1 → 6) glucochain.

How to improve the solubility of linear dextrins (LD) and retain their characteristic helix amphiphilic cavities with flexible embedding capability, is a question worth exploring without adding new chemical groups. The strategy presented in this study is to attach a highly flexible (α-1 â†’ 6) glucochain at the reducing end of LD by preparing a new type of dextrin, referred to as single-arm linear dextrin (SLD). In the actual synthesis, an (α-1 â†’ 6) linked oligosaccharide of DP¯ 10.7 (PDI = 1.28) was formed by extension of glucose units onto sucrose (2 M) by using L940W mutant of the glucansucrase GTF180-ΔN firstly. Next using γ-CD as glucosylation donor γ-CGTase extended this (α-1 â†’ 6) glucochain with (α-1 â†’ 4) bonds. SLD is a chimeric glucosaccharide comprising an (α-1 â†’ 4) linked part (DP¯ 10.5) attached to the nonreducing end of an (α-1 â†’ 6) glucochain as verified by enzyme fingerprinting and 1H NMR. Furthermore, SLD was validated to show greatly improved solubility and dispersibility of resveratrol in water, as indicated by a 3.12-fold enhancement over the solubility in the presence of 0.014 M SLD. This study provided a new strategy for solving the solubility problem of LD and opens possibilities for new design of the fine structure of starch-like materials.

Impact of Starch Binding Domain Fusion on Activities and Starch Product Structure of 4-α-Glucanotransferase.

A broad range of enzymes are used to modify starch for various applications. Here, a thermophilic 4-α-glucanotransferase from Thermoproteus uzoniensis (TuαGT) is engineered by N-terminal fusion of the starch binding domains (SBDs) of carbohydrate binding module family 20 (CBM20) to enhance its affinity for granular starch. The SBDs are N-terminal tandem domains (SBDSt1 and SBDSt2) from Solanum tuberosum disproportionating enzyme 2 (StDPE2) and the C-terminal domain (SBDGA) of glucoamylase from Aspergillus niger (AnGA). In silico analysis of CBM20s revealed that SBDGA and copies one and two of GH77 DPE2s belong to well separated clusters in the evolutionary tree; the second copies being more closely related to non-CAZyme CBM20s. The activity of SBD-TuαGT fusions increased 1.2-2.4-fold on amylose and decreased 3-9 fold on maltotriose compared with TuαGT. The fusions showed similar disproportionation activity on gelatinised normal maize starch (NMS). Notably, hydrolytic activity was 1.3-1.7-fold elevated for the fusions leading to a reduced molecule weight and higher α-1,6/α-1,4-linkage ratio of the modified starch. Notably, SBDGA-TuαGT and-SBDSt2-TuαGT showed Kd of 0.7 and 1.5 mg/mL for waxy maize starch (WMS) granules, whereas TuαGT and SBDSt1-TuαGT had 3-5-fold lower affinity. SBDSt2 contributed more than SBDSt1 to activity, substrate binding, and the stability of TuαGT fusions.

Natural antimicrobial oligosaccharides in the food industry.

An increase in the number of antibiotic resistance genes burdens the environment and affects human health. Additionally, people have developed a cautious attitude toward chemical preservatives. This attitude has promoted the search for new natural antimicrobial substances. Oligosaccharides from various sources have been studied for their antimicrobial and prebiotic effects. Antimicrobial oligosaccharides have several advantages such as being produced from renewable resources and showing antimicrobial properties similar to those of chemical preservatives. Their excellent broad-spectrum antibacterial properties are primarily because of various synergistic effects, including destruction of pathogen cell wall. Additionally, the adhesion of harmful microorganisms and the role of harmful factors may be reduced by oligosaccharides. Some natural oligosaccharides were also shown to stimulate the growth probiotic organisms. Therefore, antimicrobial oligosaccharides have the potential to meet food processing industry requirements in the future. The latest progress in research on the antimicrobial activity of different oligosaccharides is demonstrated in this review. The possible mechanism of action of these antimicrobial oligosaccharides is summarized with respect to their direct and indirect effects. Finally, the extended applications of oligosaccharides from the food source industry to food processing are discussed.

Enzymatic preparation and potential applications of agar oligosaccharides: a review.

Oligosaccharides derived from agar, that is, agarooligosaccharides and neoagarooligosaccharides, have demonstrated various kinds of bioactivities which have been utilized in a variety of fields. Enzymatic hydrolysis is a feasible approach that principally allows for obtaining specific agar oligosaccharides in a sustainable way at an industrial scale. This review summarizes recent technologies employed to improve the properties of agarase. Additionally, the relationship between the degree of polymerization, bioactivities, and potential applications of agar-derived oligosaccharides for pharmaceutical, food, cosmetic, and agricultural industries are discussed. Engineered agarase exhibited general improvement of enzymatic performance, which is mostly achieved by truncation. Rational and semi-rational design assisted by computational methods present the latest strategy for agarase improvement with greatest potential to satisfy future industrial needs. Agarase immobilized on magnetic Fe3O4 nanoparticles via covalent bond formation showed characteristics well suited for industry. Additionally, albeit with the relationship between the degree of polymerization and versatile bioactivities like anti-oxidants, anti-inflammatory, anti-microbial agents, prebiotics and in skin care of agar-derived oligosaccharides are discussed here, further researches are still needed to unravel the complicated relationship between bioactivity and structure of the different oligosaccharides.