Metabolism of Inulin via Difructose Anhydride I Pathway in Microbacterium flavum.

Difructose anhydride I (DFA-I) can be produced from inulin, with DFA-I-forming inulin fructotransferase (IFTase-I). However, the metabolism of inulin through DFA-I remains unclear. To clarify this pathway, several genes of enzymes related to this pathway in the genome of Microbacterium flavum DSM 18909 were synthesized, and the corresponding enzymes were encoded, purified, and investigated in vitro. After inulin is decomposed to DFA-I by IFTase-I, DFA-I is hydrolyzed to inulobiose by DFA-I hydrolase. Inulobiose is then hydrolyzed by ß-fructofuranosidase to form fructose. Finally, fructose enters glycolysis through fructokinase. A ß-fructofuranosidase (MfFFase1) clears the byproducts (sucrose and fructo-oligosaccharides), which might be partially hydrolyzed by fructan ß-(2,1)-fructosidase/1-exohydrolase and another fructofuranosidase (MfFFase2). Exploring the DFA-I pathway of inulin and well-studied enzymes in vitro extends our basic scientific knowledge of the energy-providing way of inulin, thereby paving the way for further investigations in vivo and offering a reference for further nutritional investigation of inulin and DFA-I in the future.

Innovative application of a novel di-D-fructofuranose 1,2':2,3'-dianhydride hydrolase (DFA-IIIase) from Duffyella gerundensis A4 to burdock root to improve nutrition.

Burdock is native to Europe and Asia and rich in many functional ingredients, including biomacromolecule polysaccharide inulin. The prebiotic fructan inulin can provide energy to organisms via several pathways. One pathway is that inulin fructotransferase (IFTase) first converts inulin to III-type difructose anhydride (DFA-III), which has many beneficial physiological functions. Then, DFA-III is hydrolyzed to inulobiose, which is a Fn-type prebiotic fructo-oligosaccharide, via difructose anhydride hydrolase (DFA-IIIase). However, there has been no study on the application of IFTase or DFA-IIIase to process burdock to increase DFA-III or inulobiose. Moreover, only five DFA-IIIases have been reported to date and all of them are from the Arthrobacter genus. Whether other microbes except for the Arthrobacter genus can utilize DFA-III through DFA-IIIase is unknown. In this work, a DFA-IIIase from Duffyella gerundensis A4 (D. gerundensis A4), abbreviated as DgDFA-IIIase, was identified and characterized in detail. DgDFA-IIIase is a bifunctional enzyme, that is, besides its hydrolytic ability to DFA-III, it has the same catalytic ability as IFTase to inulin. The enzyme was applied to the burdock root aiming at inulin and DFA-III, and inulobiose was produced with an increase in Gn-type fructo-oligosaccharide. The work verifies that microorganisms of the non-Arthrobacter genus also have the potential ability to use DFA-III by DFA-IIIase, and DFA-IIIase is feasible to increase functional substances of burdock root instead of IFTase and endo-inulinase, which paves the way for the production of functional food utilizing the polysaccharide inulin to improve nutrition and health.

Innovative application of a novel and thermostable inulin fructotransferase from Arthrobacter sp. ISL-85 to fructan inulin in burdock root to improve nutrition.

Inulin fructotransferase converts prebiotic polysaccharide inulin to difructose anhydride III, known for its numerous beneficial physiological effects. While previous studies focused on using inulin extracts under optimal conditions, this study delves into the enzyme's behavior when dealing with more complex food materials, inulin-rich burdock root, which possesses greater nutritional value but may influence the enzymatic reaction. An inulin fructotransferase from Arthrobacter sp. ISL-85 was identified and characterized, which has the highest activity of 783 U mg-1 at pH 6.5 and 65 °C and remains stable even up to 80 °C. When applied to inulin-rich burdock root (pH 4.7) at 80 °C for 2 h, the enzyme yielded 4.1 g of difructose anhydride III, concurrently increasing fructo-oligosaccharides. This study demonstrates the potential of this enzyme as a valuable tool for efficiently processing inulin within whole food materials under high temperatures. Such an approach could pave the way for enhancing nutrition and promoting health benefits.

The digestive behavior of pectin in human gastrointestinal tract: a review on fermentation characteristics and degradation mechanism.

Pectin is widely spread in nature and it develops an extremely complex structure in terms of monosaccharide composition, glycosidic linkage types, and non-glycosidic substituents. As a non-digestible polysaccharide, pectin exhibits resistance to human digestive enzymes, however, it is easily utilized by gut microbiota in the large intestine. Currently, pectin has been exploited as a novel functional component with numerous physiological benefits, and it shows a promising prospect in promoting human health. In this review, we introduce the regulatory effects of pectin on intestinal inflammation and metabolic syndromes. Subsequently, the digestive behavior of pectin in the upper gastrointestinal tract is summarized, and then it will be focused on pectin's fermentation characteristics in the large intestine. The fermentation selectivity of pectin by gut bacteria and the effects of pectin structure on intestinal microecology were discussed to highlight the interaction between pectin and bacterial community. Meanwhile, we also offer information on how gut bacteria orchestrate enzymes to degrade pectin. All of these findings provide insights into pectin digestion and advance the application of pectin in human health.

Natural edible materials made of protein-functionalized aerogel particles for postprandial hyperglycemia management.

α-Amylase inhibitors (α-AIs) delay digestion of dietary starch by inhibiting α-amylase in the gut, thereby reducing the postprandial glycemia, which is beneficial to the patients with obesity and diabetes. The proteinaceous α-AIs from wheat can effectively control starch digestion and regulate postprandial hyperglycemia. However, their gastric intolerance remains a challenge, which limits its commercial production and industrial application. In this study, sodium alginate/chitosan aerogels loaded with wheat protein α-AIs were prepared and evaluated as potential transportation and protection matrices for important components in food or pharmaceutical applications. Specifically, the biodegradable aerogel cross-linked with sodium alginate-chitosan-calcium chloride, has a large surface area and open porous structure, which can adsorb staple wheat proteins as an integrated edible material to block around 88,660 U/g of α-amylase activity. The aerogel particles were able to protect the activity of wheat α-AIs in the stomach, leading to the slow passage of the wheat α-AIs through the small intestine to inhibit starch digestion more effectively. Animal experiments further showed that the postprandial blood glucose levels in rats were effectively controlled through delayed increase, after administration of wheat protein-functionalized aerogel particles loaded with wheat α-AIs, which are natural biological macromolecules. This is a novel, safe, and economical method for the prevention and pretreatment of diabetes.

A novel hypoglycemic agent: polysaccharides from laver (Porphyra spp.).

Type-II diabetes mellitus (T2DM) has become one of the most prevalent diseases on Earth and some treatments have been developed to manage it. One intestinal enzyme α-amylase can break down starch to glucose. Inhibiting its activity will control blood glucose and provide an essential approach for the management of T2DM. Alpha-amylase inhibitor (α-AI) specifically inhibits the activity of α-amylase, and reduces the blood glucose level efficiently. To develop a novel α-AI, the red seaweed laver (Porphyra spp.) was exploited in this work, whose extracts contain polysaccharides showing an inhibitory effect against α-amylase. The crude polysaccharides were extracted using hot water (85 °C) and degraded to low-molecular-weight polysaccharides with 7% of H2O2. One polysaccharide PD-1 exhibiting a competitive binding mode with an IC50 of 12.72 mg mL-1 was separated from these degraded polysaccharides, showing approximately 98.78% of α-amylase inhibition activity. In vivo, PD-1 could efficiently suppress postprandial blood glucose levels in normal and diabetic rats. The polysaccharide inhibitor from red seaweed laver could be regarded as a novel functional food ingredient in T2DM management.

Bioavailability Based on the Gut Microbiota: a New Perspective.

The substantial discrepancy between the strong effects of functional foods and various drugs, especially traditional Chinese medicines (TCMs), and the poor bioavailability of these substances remains a perplexing problem. Understanding the gut microbiota, which acts as an effective bioreactor in the human intestinal tract, provides an opportunity for the redefinition of bioavailability. Here, we discuss four different pathways associated with the role of the gut microbiota in the transformation of parent compounds to beneficial or detrimental small molecules, which can enter the body's circulatory system and be available to target cells, tissues, and organs. We further describe and propose effective strategies for improving bioavailability and alleviating side effects with the help of the gut microbiota. This review also broadens our perspectives for the discovery of new medicinal components.

Dynamic disulfide exchange in a crystallin protein in the human eye lens promotes cataract-associated aggregation.

Increased light scattering in the eye lens due to aggregation of the long-lived lens proteins, crystallins, is the cause of cataract disease. Several mutations in the gene encoding human γD-crystallin (HγD) cause misfolding and aggregation. Cataract-associated substitutions at Trp42 cause the protein to aggregate in vitro from a partially unfolded intermediate locked by an internal disulfide bridge, and proteomic evidence suggests a similar aggregation precursor is involved in age-onset cataract. Surprisingly, WT HγD can promote aggregation of the W42Q variant while itself remaining soluble. Here, a search for a biochemical mechanism for this interaction has revealed a previously unknown oxidoreductase activity in HγD. Using in vitro oxidation, mutational analysis, cysteine labeling, and MS, we have assigned this activity to a redox-active internal disulfide bond that is dynamically exchanged among HγD molecules. The W42Q variant acts as a disulfide sink, reducing oxidized WT and forming a distinct internal disulfide that kinetically traps the aggregation-prone intermediate. Our findings suggest a redox "hot potato" competition among WT and mutant or modified polypeptides wherein variants with the lowest kinetic stability are trapped in aggregation-prone intermediate states upon accepting disulfides from more stable variants. Such reactions may occur in other long-lived proteins that function in oxidizing environments. In these cases, aggregation may be forestalled by inhibiting disulfide flow toward mutant or damaged polypeptides.

Insights into hydrolysis versus transfructosylation: Mutagenesis studies of a novel levansucrase from Brenneria sp. EniD312.

Levan is a kind of fructan that composing of fructose by ß-(2, 6) linkage and has been already applied as thickening agent and colloidal stabilizer in the cosmetic, medicinal and food industries. Microbial levansucrase is a key enzyme catalyzing the formation of levan from sucrose by transfructosylation. Here, a gene encoding levansucrase from Brenneria sp. EniD312 was cloned and expressed in Escherichia coli. The recombinant levansucrase showed the optimal pH and temperature at pH 6.5 and 45 °C. The enzyme produced 85 g/L levan from 250 g/L sucrose at pH 6.5 and 45 °C for 6 h. The residues D68, D225 and E309 of this levansucrase were speculated to be the nucleophile, the transition stabilizer and the general acid respectively by homology modelling, site-directed mutagenesis and molecular docking. Particularly, the residues in position 154 and 327 were found to play a significant role in determining the ratio of hydrolysis activity to transfructosylation activity.

Synthesis of Lactosucrose Using a Recombinant Levansucrase from Brenneria goodwinii.

Lactosucrose is a kind of trisaccharide that functions as a significant prebiotic in the maintenance of gastrointestinal homeostasis for human. In this study, a levansucrase from Brenneria goodwinii was further used for the lactosucrose production. The recombinant levansucrase showed efficiency in the lactosucrose production by transfructosylation from sucrose and lactose, and no other oligosaccharide or polysaccharide was detected in the reaction mixture. The transfructosylation product by this recombinant enzyme was structurally determined to be lactosucrose by FT-IR and NMR. The production condition was optimized as pH at 6.0, temperature at 35 °C, 5 U mL-1 enzyme, 180 g L-1 sucrose, and 180 g L-1 lactose. Under the optimal condition, the enzyme could approximately produce 100 g L-1 lactosucrose when the reaction reached equilibrium. The recombinant levansucrase could effectively and exclusively catalyze the formation of lactosucrose, which might expand the enzymatic choice for further preparation of lactosucrose.

Recent advances on biological production of difructose dianhydride III.

Difructose dianhydride III (DFA III) is a cyclic difructose containing two reciprocal glycosidic linkages. It is easily generated with a small amount by sucrose caramelization and thus occurs in a wide range of food-stuffs during food processing. DFA III has half sweetness but only 1/15 energy of sucrose, showing potential industrial application as low-calorie sucrose substitute. In addition, it displays many benefits including prebiotic effect, low cariogenicity property, and hypocholesterolemic effect, and improves absorption of minerals, flavonoids, and immunoglobulin G. DFA III is biologically produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). Plenty of DFA III-producing enzymes have been identified. The crystal structure of inulin fructotransferase has been determined, and its molecular modification has been performed to improve the catalytic activity and structural stability. Large-scale production of DFA III has been studied by various IFTases, especially using an ultrafiltration membrane bioreactor. In this article, the recent findings on physiological effects of DFA III are briefly summarized; the research progresses on identification, expression, and molecular modification of IFTase and large-scale biological production of DFA III by IFTase are reviewed in detail.

Physicochemical properties of a high molecular weight levan from Brenneria sp. EniD312.

A high molecular weight levan was produced by a novel levansucrase and some properties of this polymer were investigated. The levan exhibited a poroid microstructure as well as series of individual ellipsoidal or spheroidal particles. The weight-average molecular weight (M¯w) of the levan was determined to be 1.41×108Da. In a 0.1% solution, the levan showed a mean diameter of 176nm, while in a 1% solution the diameter was 182nm. The decomposition temperature was determined to be 216.67°C, with an endothermic peak at 147.41°C and a melting enthalpy of 76.9J/g. The small angle X-ray diffraction pattern showed a distinctive peak pattern between 15° and 40° (2q). The levan solution showed a shear-thinning behaviour. These results suggest this levan could be a good additive in the food processing industry, as well as an important bio-based material in the medicinal or chemical industry.

Improving the Catalytic Behavior of DFA I-Forming Inulin Fructotransferase from Streptomyces davawensis with Site-Directed Mutagenesis.

Previously, a α-d-fructofuranose-ß-d-fructofuranose 1,2':2,1'-dianhydride (DFA I)-forming inulin fructotransferase (IFTase), namely, SdIFTase, was identified. The enzyme does not show high performances. In this work, to improve catalytic behavior including activity and thermostability, the enzyme was modified using site-directed mutagenesis on the basis of structure. The mutated residues were divided into three groups. Those in group I are located at central tunnel including G236, A257, G281, T313, and A314S. The group II contains residues at the inner edge of substrate binding pocket including I80, while group III at the outer edge includes G121 and T122. The thermostability was reflected by the melting temperature (Tm) determined by Nano DSC. Finally, the Tm values of G236S/G281S/A257S/T313S/A314S in group I and G121A/T122L in group III were enhanced by 3.2 and 4.5 °C, and the relative activities were enhanced to 140.5% and 148.7%, respectively. The method in this work may be applicable to other DFA I-forming IFTases.

Enzymatic Production of Melibiose from Raffinose by the Levansucrase from Leuconostoc mesenteroides B-512 FMC.

Melibiose, which is an important reducing disaccharide formed by α-1,6 linkage between galactose and glucose, has been proven to have beneficial applications in both medicine and agriculture. In this study, a characterized levansucrase from Leuconostoc mesenteroides B-512 FMC was further used to study the bioproduction of melibiose from raffinose. The reaction conditions were optimized for melibiose synthesis. The optimal pH, temperature, substrate concentration, ratio of substrates, and units of enzymes were determined as pH 6.0, 45 °C, 210 g/L, 1:1 (210 g/L:210 g/L), and 5 U/mL, respectively. The transfructosylation product of raffinose was determined to be melibiose by FTIR and NMR. A high raffinose concentration was found to strongly favor the production of melibiose. When 210 g/L raffinose and 210 g/L lactose were catalyzed using 5 U/mL purified levansucrase at pH 6.0 and 45 °C, the maximal yield of melibiose was 88 g/L.

Efficient biosynthesis of levan from sucrose by a novel levansucrase from Brenneria goodwinii.

Levan, a unique homopolysaccharide consisting of fructose residues linked by ß-(2, 6) bonds, possess promising physiochemical and physiological properties with numerous potential applications. In this study, a novel levan-producing levansucrase was characterized from Brenneria goodwinii. The polysaccharide produced by the recombinant enzyme from sucrose was structurally determined to be levan-type fructan connected by ß-(2, 6) linkages. The optimum pH was measured to be pH 6.0 for both sucrose hydrolysis and transfructosylation. The optimum temperature was 35, 45, and 40°C for transfructosylation, sucrose hydrolysis, and total activity, respectively. Higher sucrose concentration greatly favored to levan biosynthesis. The purified recombinant enzyme produced 185g/L levan from 50% (w/v) sucrose at pH 6.0 and 35°C for 12h. The molecular weight of the produced levan polysaccharide reached 1.3×108Da, which was much higher than the ones produced by many reported levansucrases.

Synthesis of raffinose by transfructosylation using recombinant levansucrase from Clostridium arbusti SL206.

BACKGROUND: Raffinose, a functional trisaccharide of α-d-galactopyranosyl-(1 → 6)-α-d-glucopyranosyl-(1 → 2)-ß-d-fructofuranoside, is a prebiotic that shows promise for use as a food ingredient. RESULTS: In this study, the production of raffinose from melibiose and sucrose was studied using whole recombinant Escherichia coli cells harboring the levansucrase from Clostridium arbusti SL206. The reaction conditions were optimized for raffinose synthesis. The optimal pH, temperature and washed cell concentration were pH 6.5 (sodium phosphate buffer, 50 mmol L-1 ), 55 °C and 3% (w/v), respectively. High substrate concentrations, which led to low water activity and thus reduced levansucrase hydrolysis activity, strongly favored the production of raffinose through the fructosyl transfer reaction. Additionally, high concentrations of excess acceptor and donor glycosides favored raffinose production. When 30% (w/v) sucrose and 30% (w/v) melibiose were catalyzed using 3% (w/v) whole cells at pH 6.5 (sodium phosphate buffer, 50 mmol L-1 ) and 55 °C, the highest raffinose yield was 222 g L-1 after a 6 h reaction. The conversion ratio from each substrate to raffinose was 50%. CONCLUSION: Raffinose could be effectively produced with melibiose as an acceptor and with sucrose as a fructosyl donor by whole recombinant E. coli cells harboring C. arbusti levansucrase. The yield from E. coli was significantly higher than those of the previously reported Bacillus subtilis levansucrase and fungal α-galactosidases. © 2016 Society of Chemical Industry.

Identification of a novel DFA I-producing inulin fructotransferase from Streptomyces davawensis.

In this work, a novel gene encoding DFA I-forming inulin fructotransferase (IFTase) from Streptomyces davawensis SK39.001 was cloned and expressed in Escherichia coli. The enzyme was purified, identified, and characterized. The results showed that this IFTase (DFA I-forming) is a trimer (molecular weight of 125KDa) consisting of three identical subunits (the molecular weight as assayed by SDS-PAGE was approximately 40KDa). At pH 5.5 and 40°C, the maximum specific activity (approximately 100Umg-1) was achieved. Moreover, the enzyme was stable up to 70°C. Km and Vmax were 2.89±0.2mM and 1.94±0.9mMmin-1, respectively. For exploring putative active sites and probable catalytic mechanisms, homology modelling and molecular docking methods after site-directed mutagenesis were applied to IFTase (DFA I-forming). The results revealed that D183 and E194 were potential catalytic residues of the purified enzyme.

Probing the Role of Two Critical Residues in Inulin Fructotransferase (DFA III-Producing) Thermostability from Arthrobacter sp. 161MFSha2.1.

Inulin fructotransferase (IFTase) is an important enzyme that produces di-d-fructofuranose 1,2':2,3' dianhydride (DAF III), which is beneficial for human health. Present investigations mainly focus on screening and characterizing IFTase, including catalytic efficiency and thermostability, which are two important factors for enzymatic industrial applications. However, few reports aimed to improve these two characteristics based on the structure of IFTase. In this work, a structural model of IFTase (DFA III-producing) from Arthrobacter sp. 161MFSha2.1 was constructed through homology modeling. Analysis of this model reveals that two residues, Ser-309 and Ser-333, may play key roles in the structural stability. Therefore, the functions of the two residues were probed by site-directed mutagenesis combined with the Nano-DSC method and assays for residual activity. In contrast to other mutations, single mutation of serine 309 (or serine 333) to threonine did not decrease the enzymatic stability, whereas double mutation (serine 309 and serine 333 to threonine) can enhance thermostability (by approximately 5 °C). The probable mechanisms for this enhancement were investigated.

Improving the Thermostability and Catalytic Efficiency of the d-Psicose 3-Epimerase from Clostridium bolteae ATCC BAA-613 Using Site-Directed Mutagenesis.

d-Psicose is a highly valuable rare sugar because of its excellent physiological properties and commercial potential. d-Psicose 3-epimerase (DPEase) is the key enzyme catalyzing the isomerization of d-fructose to d-psicose. However, the poor thermostability and low catalytic efficiency are serious constraints on industrial application. To address these issues, site-directed mutagenesis of Tyr68 and Gly109 of the Clostridium bolteae DPEase was performed. Compared with the wild-type enzyme, the Y68I variant displayed the highest substrate-binding affinity and catalytic efficiency, and the G109P variant showed the highest thermostability. Furthermore, the double-site Y68I/G109P variant was generated and exhibited excellent enzyme characteristics. The Km value decreased by 17.9%; the kcat/Km increased by 1.2-fold; the t1/2 increased from 156 to 260 min; and the melting temperature (Tm) increased by 2.4 °C. Moreover, Co(2+) enhanced the thermostability significantly, including the t1/2 and Tm values. All of these indicated that the Y68I/G109P variant would be appropriate for the industrial production of d-psicose.

Identification of a Novel Di-D-Fructofuranose 1,2':2,3' Dianhydride (DFA III) Hydrolysis Enzyme from Arthrobacter aurescens SK8.001.

Previously, a di-D-fructofuranose 1,2':2,3' dianhydride (DFA III)-producing strain, Arthrobacter aurescens SK8.001, was isolated from soil, and the gene cloning and characterization of the DFA III-forming enzyme was studied. In this study, a DFA III hydrolysis enzyme (DFA IIIase)-encoding gene was obtained from the same strain, and the DFA IIIase gene was cloned and expressed in Escherichia coli. The SDS-PAGE and gel filtration results indicated that the purified enzyme was a homotrimer holoenzyme of 145 kDa composed of subunits of 49 kDa. The enzyme displayed the highest catalytic activity for DFA III at pH 5.5 and 55°C, with specific activity of 232 U mg-1. Km and Vmax for DFA III were 30.7 ± 4.3 mM and 1.2 ± 0.1 mM min-1, respectively. Interestingly, DFA III-forming enzymes and DFA IIIases are highly homologous in amino acid sequence. The molecular modeling and docking of DFA IIIase were first studied, using DFA III-forming enzyme from Bacillus sp. snu-7 as a template. It was suggested that A. aurescens DFA IIIase shared a similar three-dimensional structure with the reported DFA III-forming enzyme from Bacillus sp. snu-7. Furthermore, their catalytic sites may occupy the same position on the proteins. Based on molecular docking analysis and site-directed mutagenesis, it was shown that D207 and E218 were two potential critical residues for the catalysis of A. aurescens DFA IIIase.