Cyclodextrin Glycosyltransferase Research Articles

Improving cyclodextrin yield via retarding retrogradation of debranched starches during the preparation of cyclodextrin with cyclodextrin glycosyltransferase and pullulanase

Improving substrate utilization is crucial for industrial conversion of starch. The introduction of pullulanase in cyclodextrin (CD) production has improved substrate utilization. However, complete utilization of debranched starches has not been achieved, mainly due to the retrogradation of debranched starches and the inhibitory effect of CDs on pullulanase during the catalytic reaction. This study aimed to establish an efficient CD preparation system by regulating the substrate concentration and reaction temperature. A Bacillus thermoleovorans pullulanase (BtPul) was selected for investigation of its interaction with CDs through inhibition pattern assays, fluorescence spectroscopy, and molecular docking. Results clarified that CDs inhibited BtPul, with α-CD showing the highest inhibition, followed by β-CD and γ-CD. γ-CD inhibited BtPul through a mixed mode of competitive and noncompetitive inhibition, while the other CDs inhibited BtPul competitively. When pullulan was used as the substrate, increasing the substrate concentration weakened the inhibitory effect of CDs to some degree, while changes in reaction temperature only had a minor contribution. When BtPul and β-CGTase collaboratively converted 30% (w/v) starch substrate, an increase in reaction temperature improved the conversion rate of β-CD from 67.6% to 86.8%. Optimization of substrate concentration showed that high concentration starch substrate had an adverse effect on CDs yield due to retrogradation. The optimal yields of α-CD and β-CD produced by BtPul and α-/β-CGTases were 55.7% and 90.3%, respectively. This study provides a reliable method and reference for the efficient conversion of starch into CDs.

Enhancement of β-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 β-CD by hydrolyzing α-1,6 glucosidic bonds. The addition of SsGDE (750 U/g of starch) at the liquefaction stage remarkably improved the β-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 β-CGTase to convert into CDs. Overall, these findings proposed a new approach of combining SsGDE with β-CGTase to enhance the CD yield.

Immobilization of Cyclodextrin glycosyltransferase onto three dimensional- hydrophobic and two dimensional- hydrophilic supports: A comparative study.

Cyclodextrin glycosyltransferase (CGTase) degrades starch into cyclodextrin via enzymatic activity. In this study, we immobilize CGTase from Thermoanaerobacter sp. on two supports, namely graphene nanoplatelets (GNP) consisting of short stacks of graphene nanoparticles and a calcium-based two-dimensional metal organic framework (Ca-TMA). The uptakes of CGTase on GNP and Ca-TMA reached 40 and 21mg g-1 respectively, but immobilized CGTase on Ca-TMA showed a higher specific activity (38 U mg-1 ) than that on GNP (28 U mg-1 ). Analysis of secondary structures of CGTase, shows that immobilization reduces the proportion of β-sheets in CGTase from 56% in the free to 49% and 51.3% for GNP and Ca-TMA respectively, α-helix from 38.5% to 18.1 and 37.5%, but led to increased β-turns from 5.5 to 40% and 11.2% for GNP and Ca-TMA, respectively. Lower levels of conformational changes were observed over the more hydrophilic Ca-TMA compared to hydrophobic GNP, resulting in its better activity. Increased β-turns were found to correlate with lower β-CD production, while more β-sheets and α-helix favored more β-CD. Reusability studies revealed that GNP retains up to 74% of initial CGTase activity, while Ca-TMA dropped to 33% after eight consecutive uses. The results obtained in this work provide insight on the effect of support's surface properties on CGTase performance and can assist in developing robust CGTase-based biocatalysts for industrial application.

Surface engineering of cyclodextrin glycosyltransferase reveals structural compactness and rigidity responsible for enhanced organic solvents resistance

Cyclodextrin glycosyltransferase (CGTase) is a preferable biocatalyst for production of cyclodextrin and phenols glycosylated derivatives due to its cost-effective donors and broad range of acceptors. Organic solvents (OSs) are often beneficial for solubilizing hydrophobic substrates and product specificity, but often harmful towards CGTases. Herein, using Paenibacillus macerans CGTase (PmCGTase) as a template and DMSO as a screening solvent, semi-rational engineering was performed at 18 surface residues by a developed high throughput screening method. Using site saturation mutagenesis and iterative saturation mutagenesis, 5 OSs-resistant variants (G539I, G539V, G539I/R146F, G539V/R146A, G539I/R146F/D147N) were obtained. Compared with WT, the best variant G539I/R146F/D147N showed a 79.3 % enhanced residual activity at 30 % DMSO and a 60 % increased t1/2 value at both 40 and 50 °C. In addition, a decreased Km value and an increased kcat value led to 41.9 % higher catalytic efficiency of G539I/R146F/D147N than WT in 15 % DMSO. This variant was also applied to glycosylation of sophoricoside in 20 % DMSO, leading to 26.2 % increased conversion. Molecular dynamics demonstrates that its enhanced OSs resistance may be attributed to the strengthened structural compactness and rigidity. Our results provide guidance for engineering OSs resistance of PmCGTase, which would further improve its application potential.

Bioconversion of starch to Cyclodextrin using Cyclodextrin glycosyltransferase immobilized on metal organic framework

The superiority of a metal organic framework, namely, MIL-101 (Cr) over conventional support, zeolite Y, as immobilization support for Cyclodextrin glycosyltransferase (CGTase) was investigated. The adsorption capacity, stability, and secondary structures of CGTase upon immobilization were investigated. CGTase adsorption on MIL-101 and zeolite Y was best represented by the Langmuir isotherm with optimum adsorption capacities of 37.5 and 6.1 mg/g on two supports, respectively. The deconvolution of the amide I band of the FTIR spectrum indicated that free CGTase molecules predominantly contain β-sheets, which increased with the immobilization on MIL-101 and zeolite Y from 56% to 84.1% and 69.7%, respectively, suggesting the existence of CGTase agglomerates on the supports. The CGTase immobilized on MIL-101 showed relative activity of about 50% at a substrate concentration of up to 4 g/L (compared with free enzymes) and above 3 times higher selectivity towards the more valuable α-CD. A diffusion–reaction model was used to predict the behavior of the immobilized system, and reusability studies revealed that immobilized CGTase may be utilized for numerous reaction cycles, with possible improvements via covalent attachment using glutaraldehyde. Overall, the obtained results in this study provide insights into the usefulness of MIL-101 as a suitable support for CGTase.

Economical synthesis of γ-cyclodextrin catalyzed by oriented cyclodextrin glycosyltransferase displayed on bacterial polyhydroxyalkanoate nanogranules

BackgroundThe advantages of γ-cyclodextrin (γ-CD) include its high solubility, ability to form inclusion complexes with various poorly water-soluble molecules, and favorable toxicological profile; thus, γ-CD is an attractive functional excipient widely used in many industrial settings. Unfortunately, the high cost of γ-CD caused by the low activity and stability of γ-cyclodextrin glycosyltransferase (γ-CGTase) has hampered large-scale production and application.ResultsThis study reports the in vivo one-step production of immobilized γ-CGTase decorated on the surface of polyhydroxyalkanoate (PHA) nanogranules by the N-terminal fusion of γ-CGTase to PHA synthase via a designed linker. The immobilized γ-CGTase-PHA nanogranules showed outstanding cyclization activity of 61.25 ± 3.94 U/mg (γ-CGTase protein) and hydrolysis activity of 36,273.99 ± 1892.49 U/mg, 44.74% and 18.83% higher than that of free γ-CGTase, respectively. The nanogranules also exhibited wider optimal pH (cyclization activity 7.0–9.0, hydrolysis activity 10.0–11.0) and temperature (55–60 °C) ranges and remarkable thermo- and pH-stability, expanding its utility to adapt to wider and more severe reaction conditions than the free enzyme. A high yield of CDs (22.73%) converted from starch and a high ratio (90.86%) of γ-CD in the catalysate were achieved at pH 9.0 and 50 °C for 10 h with 1 mmol/L K+, Ca2+, and Mg2+ added to the reaction system. Moreover, γ-CGTase-PHA beads can be used at least eight times, retaining 82.04% of its initial hydrolysis activity and 75.73% of its initial cyclization activity.ConclusionsThis study provides a promising nanobiocatalyst for the cost-efficient production of γ-CD, which could greatly facilitate process control and economize the production cost.

Engineering of cyclodextrin glycosyltransferase improves the conversion efficiency of rebaudioside A to glucosylated steviol glycosides and increases the content of short-chain glycosylated steviol glycoside

BackgroundCompared with steviol glycosides, the taste of glucosylated steviol glycosides is better and more similar to that of sucrose. At present, cyclodextrin glucanotransferase (CGTase) is primarily used to catalyze the conversion of steviol glycosides to glucosylated steviol glycosides, with soluble starch serving as a glycosyl donor. The main disadvantages of enzymatic transglycosylation are the limited number of enzymes available, the low conversion rates that result in low yields, and the lack of selectivity in the degree of glycosylation of the products. In order to fill these gaps, the proteome of Alkalihalobacillus oshimensis (also named Bacillus oshimensis) was used for mining novel CGTases.ResultsHere, CGTase-15, a novel β-CGTase with a wide pH adaptation range, was identified and characterized. The catalyzed product of CGTase-15 tasted better than that of the commercial enzyme (Toruzyme® 3.0 L). In addition, two amino acid sites, Y199 and G265, which play important roles in the conversion of steviol glycosides to glucosylated steviol glycosides were identified by site-directed mutagenesis. Compared with CGTase-15, CGTase-15-Y199F mutant significantly increased the conversion rate of rebaudioside A (RA) to glucosylated steviol glycosides. Compared with CGTase-15, the content of short-chain glycosylated steviol glycosides catalyzed by CGTase-15-G265A mutant was significantly increased. Moreover, the function of Y199 and G265 was verified in other CGTases. The above mutation pattern has also been applied to CGTase-13 (a CGTase discovered by our laboratory with great potential in the production of glycosylated steviol glycosides), confirming that the catalytic product of CGTase-13-Y189F/G255A mutant has a better taste than that of CGTase-13.ConclusionsThis is the first report on the improvement of the sensory profiles of glycosylated steviol glycosides through site-directed mutagenesis of CGTase, which is significant for the production of glycosylated steviol glycosides.

Rational Design of Cyclodextrin Glycosyltransferase with Improved Hesperidin Glycosylation Activity

Cyclodextrin glycosyltransferase (CGTase) can catalyze the glycosylation of hesperidin, resulting in α-glycosyl hesperidin with significantly improved water solubility. In this study, a rational design of CGTase to improve its hesperidin glycosylation activity was investigated. The strategy we employed involved docking hesperidin in its near-attack conformation and virtually mutating the surrounding residues, followed by calculating the changes in binding energy using Rosetta flex-ddG. The mutations with a stabilization effect were then subjected to an activity assay. Starting from CGTase-Y217F, we obtained three double-point mutants, Y217F/M351F, Y217F/M351L, and Y217F/D393H, with improved hesperidin glycosylation activities after screening twenty variants. The best variant, Y217F/D393H, exhibited a catalytic activity of 1305 U/g, and its kcat/KmA is 2.36 times higher compared to CGTase-Y217F and 15.14 times higher compared to the wild-type CGTase. Molecular dynamic simulations indicated that hesperidin was repulsed by CGTase-Y217F when bound in a near-attack conformation. However, by introducing a second-point mutation with a stabilization effect, the repulsion effect is weakened, resulting in a reduction in the distances between the bond-forming atoms and, thus, favoring the reaction.

Enhancing 2-O-α-D-glucopyranosyl-L-ascorbic acid synthesis by weakening the acceptor specificity of CGTase toward glucose and maltose.

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid (L-AA), which has been widely used in food and cosmetics industries. Sugar molecules, such as glucose and maltose produced by cyclodextrin glycosyltransferase (CGTase) during AA-2G synthesis may compete with L-AA as the acceptors, resulting in low AA-2G yield. Multiple sequence alignment combined with structural simulation analysis indicated that residues at positions 191 and 255 of CGTase may be responsible for the difference in substrate specificity. To investigate the effect of these two residues on the acceptor preference and the AA-2G yield, five single mutants Bs F191Y, Bs F255Y, Bc Y195F, Pm Y195F and Pm Y260F of three CGTases from Bacillus stearothermophilus NO2 (Bs), Bacillus circulans 251 (Bc) and Paenibacillus macerans (Pm) were designed for AA-2G synthesis. Under optimal conditions, the AA-2G yields of the mutants Bs F191Y and Bs F255Y AA-2G were 34.3% and 7.9% lower than that of Bs CGTase, respectively. The AA-2G yields of mutant Bc Y195F, Pm Y195F and Pm Y260F were 45.8%, 36.9% and 12.6% higher than those of wild-type CGTases, respectively. Kinetic studies revealed that the residues at positions 191 and 255 of the three CGTases were F, which decreased glucose and maltose specificity and increased L-AA specificity. This study not only proposes for the first time that the AA-2G yield can be improved by weakening the acceptor specificity of CGTase toward sugar byproducts, but also provides new insight on the modification of CGTase that catalyze the double-substrate transglycosylation reaction.

Heterologous expression of cyclodextrin glycosyltransferase from Bacillus stearothermophilus in Bacillus subtilis and its application in glycosyl rutin production.

The online version contains supplementary material available at 10.1007/s13205-023-03510-5.

Aided-efflux and high production of β-amyrin realized by β-cyclodextrin in situ synthesized on surface of Saccharomyces cerevisiae.

As a plant-derived pentacyclic triterpenoid, β-amyrin has been heterogeneously synthesized in Saccharomyces cerevisiae. However, β-amyrin is intracellularly produced in a lower gram scale using recombinant S. cerevisiae, which limits the industrial applications. Although many strategies have been proven to be effective to improve the production of β-amyrin, the intracellularly accumulation is still a challenge in reaching higher titer and simplifying the extraction process. To solve this problem, the amphiphilic β-cyclodextrin (β-CD) has been previously employed to aid the efflux of β-amyrin out of the cells. Nevertheless, the supplemented β-CD in the medium is not consistent with β-amyrin synthesis and has the disadvantage of rather high cost. Therefore, an aided-efflux system based on in situ synthesis of β-CD was developed in this study to enhance the biosynthesis of β-amyrin and its efflux. The in situ synthesis of β-CD was started from starch by the surface displayed cyclodextrin glycosyltransferase (CGTase) on yeast cells. As a result, the synthesized β-CD could capture 16% of the intracellular β-amyrin and improve the total production by 77%. Furthermore, more strategies including inducing system remodeling, precursor supply enhancement, two-phase fermentation and lipid synthesis regulation were employed. Finally, the production of β-amyrin was increased to 73 mg/L in shake flask, 31 folds higher than the original strain, containing 31 mg/L of extracellular β-amyrin. Overall, this work provides novel strategies for the aided-efflux of natural products with high hydrophobicity in engineered S. cerevisiae.

Glycosyltransferases improve breadmaking quality by altering multiscale structure in gluten-free bread

In gluten-free bread (GFB), starch and its interplay with structural agents were vital for product quality due to the absence of gluten. During breadmaking, amylolytic enzymes modified starch in situ , and their reaction modes determined starch structure and hence how interactions occurred. Herein, based on the matrices of rice protein and hydroxypropyl methylcellulose (HPMC), α-amylase and multifunctional enzymes of glucan branching enzyme (GBE) and cyclodextrin glycosyltransferases (CGTase) were adopted, aiming to understand the mechanism of these enzymes on breadmaking quality of fresh bread from the multiscale structure of GFB. Results demonstrated that all enzymes, especially CGTase and GBE, markedly increased GFB specific volume to 107%–134% of Control. Compared with α-amylase, CGTase and GBE more depended on the structural changes rather than fermentation to improve GFB volume. Considering the texture, CGTase and GBE reduced crumb stickiness and still maintained a relatively softness and springiness crumb, while α-amylase induced a stick and less elastic crumb. Starch molecular changes enhanced the entanglement of starch and HPMC to form extended and bold fibril bundles and finally improved strength and pore size of gel network structure. Moreover, the starch structure modifications and specific enzymatic products of CGTase and GBE altered the microstructure and thus facilitated the even distribution of rice protein, which further improved GFB texture properties. Amylolytic enzymes, that acted both hydrolysis and glycosyl transfer, moderately hydrolyzed starch and generated specific enzymatic products, were more suitable to enhance GFB structure and eliminate the weak crumb texture. • α-amylase (α-Amy) and glycosyltransferases (GTases) were included. • Breadmaking quality of gluten-free bread (GFB) with α-Amy and GTases was compared. • GFB with GTases performed better in crumb structure and texture. • GTases depended more on structure rather than gas to improve volume. • Interactions of enzymatic starch and structural agents was crucial for GFB quality.

Novel α-glycosyl compounds from glycosylation of rubusoside

In this work we investigated mixtures from α-glycosylation of rubusoside with cyclodextrin glycosyltransferases. In addition to the previously known α-1,4 glycosylated derivatives, nine new compounds with rare α-1,3-glycosidic bonds were identified based on nuclear magnetic resonance spectroscopy and mass spectrometric analysis. Furthermore, sensory properties of monoglycosylated rubusoside derivatives were investigated and compared to previously described monoglycosylated compounds. Additionally, digestion with α-amylase from human saliva was investigated for different glycosylated rubusoside derivatives.

Microbial Cyclodextrin Glycosyltransferases: Sources, Production, and Application in Cyclodextrin Synthesis

Cyclodextrin glycosyltransferase (CGTase) is a multifunctional enzyme that hydrolyzes the <em>α</em>-glycosidic bond between two sugar molecules and synthesizes cyclodextrins (CDs) and other transglycosylation products. It is a ubiquitously present extracellular enzyme that offers the CGTase-producing organism the sole right onto starch substrates over other microbes. The present review provides a brief account of diversity among CGTase-producing microbes, CGTase production in different heterologous hosts (wherein extracellular secretion is highly desired), and different physicochemical properties of CGTases. Overall, 52 crystal structures that highlight the five domain tertiary structure of CGTases have been discovered so far. On the basis of these structures, the catalytic mechanism of CGTase reactions has been discussed, and three catalytic residues, namely Glu257, Asp229, and Asp328, have been identified at the active site in all CGTases. Moreover, the active site is constituted by at least nine sugar-binding sites, denoted as -7 to +2. Furthermore, a sequence alignment of selected CGTases highlighted the conserved regions and the sequential differences among <em>α</em>-CGTases, <em>β</em>-CGTases, and <em>γ</em>-CGTases. Various biotechnological applications of CGTases and CGTase immobilization on a variety of support matrices are briefly discussed. This review also encompasses a detailed account of CDs, their enzymatic production, extraction, and applications in different industrial sectors.

Genetic enhancement of Bacillus cyclodextrin glycosyltransferase production

Genetic enhancement of Bacillus cyclodextrin glycosyltransferase production

Molecular mechanism of acceptor selection in cyclodextrin glycosyltransferases catalyzed ginsenoside transglycosylation

Understanding the mechanisms of enzyme specificity is increasingly important from a fundamental viewpoint and for practical applications. Transglycosylation has attracted many attentions due to its importance in improving the functional properties of acceptor substrates both in vivo and in vitro. Cyclodextrin glucanotransferase (CGTase) is one of the key enzymes in transglycosylation, it has a broad substrate spectrum and utilizes sugar as the donor. However, little is known about the acceptor selectivity of CGTase, which greatly hampers efforts toward the rational design of desirable transglycosylated derivatives. In this study, we found that the CGTase from Bacillus circulans, BcCGTase, was able to form glycosylated products with diverse ginsenosides. In particular, it not only carries out diverse mono-, di-, and even higher-order glycosylations via the transfer of glucose moieties to the COGlc positions, but also can glycosylate the C3-OH position of ginsenosides. In contrast, another CGTase from Bacillus licheniformis (BlCGTase) showed relatively specific acceptor preference with only several ginsenosides. Structural comparison between BcCGTase and BlCGTase revealed that the Arg74/K81 position within the acceptor-binding sites of BcCGTase/BlCGTase was responsible for the differences in catalytic specificity for ginsenoside F1. Further mutagenesis confirmed their roles in the acceptor selection. In conclusion, our study not only demonstrates the acceptor selectivity of CGTases, but also provides insight into the catalytic mechanism of CGTases, which will potentially increase the utility of CGTase for biosynthesis of new, rationally designed transglycosylated derivatives.

Engineering of Cyclodextrin Glycosyltransferase through a Size/Polarity Guided Triple-Code Strategy with Enhanced α-Glycosyl Hesperidin Synthesis Ability.

Hesperidin, a flavonoid enriched in citrus peel, can be enzymatically glycosylated using CGTase with significantly improved water solubility. However, the reaction catalyzed by wild-type CGTase is rather inefficient, reflected in the poor production rate and yield. By focusing on the aglycon attacking step, seven residues were selected for mutagenesis in order to improve the transglycosylation efficiency. Due to the lack of high-throughput screening technology regarding to the studied reaction, we developed a size/polarity guided triple-code strategy in order to reduce the library size. The selected residues were replaced by three rationally chosen amino acids with either changed size or polarity, leading to an extremely condensed library with only 32 mutants to be screened. Twenty-five percent of the constructed mutants were proved to be positive, suggesting the high quality of the constructed library. Specific transglycosylation activity of the best mutant Y217F was assayed to be 935.7 U/g, and its kcat/KmA is 6.43 times greater than that of the wild type. Homology modeling and docking computation suggest the source of notably enhanced catalytic efficiency is resulted from the combination of ligand transfer and binding effect. IMPORTANCE Size/polarity guided triple-code strategy, a novel semirational mutagenesis strategy, was developed in this study and employed to engineer the aglycon attacking site of CGTase. Screening pressure was set as improved hesperidin glucoside synthesis ability, and eight positive mutants were obtained by screening only 32 mutants. The high quality of the designed library confirms the effectiveness of the developed strategy is potentially valuable to future mutagenesis studies. Mechanisms of positive effect were explained. The best mutant exhibits 6.43 times enhanced kcat/KmA value and confirmed to be a superior whole-cell catalyst with potential application value in synthesizing hesperidin glucosides.

Fisetin glycosides synthesized by cyclodextrin glycosyltransferase from Paenibacillus sp. RB01: characterization, molecular docking, and antioxidant activity.

Fisetin is a flavonoid that exhibits high antioxidant activity and is widely employed in the pharmacological industries. However, the application of fisetin is limited due to its low water solubility. In this study, glycoside derivatives of fisetin were synthesized by an enzymatic reaction using cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 in order to improve the water solubility of fisetin. Under optimal conditions, CGTase was able to convert more than 400 mg/L of fisetin to its glycoside derivatives, which is significantly higher than the previous biosynthesis using engineered E. coli. Product characterization by HPLC and LC-MS/MS revealed that the transglycosylated products consisted of at least five fisetin glycoside derivatives, including fisetin mono-, di- and triglucosides, as well as their isomers. Enzymatic analysis by glucoamylase and α-glucosidase showed that these fisetin glycosides were formed by α-1,4-glycosidic linkages. Molecular docking demonstrated that there are two possible binding modes of fisetin in the enzyme active site containing CGTase-glysosyl intermediate, in which O7 and O4’ atoms of fisetin positioned close to the C1 of glycoside donor, corresponding to the isomers of the obtained fisetin monoglucosides. In addition, the water solubility and the antioxidant activity of the fisetin monoglucosides were tested. It was found that their water solubility was increased at least 800 times when compared to that of their parent molecule while still maintaining the antioxidant activity. This study revealed the potential application of CGTase to improve the solubility of flavonoids.

Enhancement of the water solubility and antioxidant capacities of mangiferin by transglucosylation using a cyclodextrin glycosyltransferase

This study aimed to enhance the water solubility and antioxidant properties of mangiferin by transglucosylation using cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. The highest mangiferin to mangiferin glucoside conversion yield achieved was 88.9% using 60 mU/mL CGTase, 25 mM mangiferin, and 10% starch (w/v), with incubation at 60 °C for 10 h. The product of transglucosylation was purified and its chemical structure was determined to be glucosyl-α-(1→4)-mangiferin (MGF-g1) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. The water solubility of MGF-g1 was 5,093 times higher than that of mangiferin. MGF-g1 exhibited 1.6-fold higher 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, 1.24-fold higher oxygen radical antioxidant capacity, and 1.19-fold higher ferric reducing ability power, compared to mangiferin. Moreover, the cyclooxygenase-2 inhibitory activity (IC50) of mangiferin and MGF-g1 were 76.44 ± 11.7 μM and 59.74 ± 2.8 μM, respectively. Our results suggest that the novel MGF-g1 has potential applications as a functional material in the food and pharmaceutical industries.

Bio-enhancement of Soy Isoflavones (Genistein & Daidzein) Using Bacillus coagulans in Letrozole Induced Polycystic Ovarian Syndrome by Regulating Endocrine Hormones in Rats.

Soy isoflavone (SIF), a natural phytoestrogen, is used in the condition of hormonal imbalance. These isoflavones generally have low solubility resulting in low bioavailability and bioactivity. It is reported that trans-glycosylation by cyclodextrin glycosyltransferase (CGTase) is widely utilized for increasing the solubility and bioavailability of isoflavones. Present investigation was aimed to study the effect of Bacillus coagulans (a probiotic) in potentiating the bioactivity of soy isoflavones in letrozole-induced PCOS. Initial consideration was focused on proving CGTase assay of B. coagulans. After that, animal study was performed to check the enhancement of bioactivity of SIF along with B. coagulans. A total of 36 rats, separated into six groups (6 rats in each), were used. Group I received vehicles, group II received letrozole (1mg/kg) for 21days, and group III animals were administered with soy isoflavones (SIF-100mg/kg). In the case of group IV, V, and VI, animals received SIF (100mg/kg) along with B. coagulans 0.65, 3.25, and 6.50mg/kg, respectively. Treatment was given for 2weeks after induction of disease. All the animals were sacrificed at the end of the study and endpoint parameters were performed. Present investigation revealed that combination of SIF with B. coagulans showed hormone restoration, reduce oxidative stress, recovery in the menstrual cycle, and improvement in ovarian physiology. SIF (genistein & daidzein) together withB. coagulansexhibits a beneficial role in the enhancement of the bioactivity of soy isoflavones.Further, it showed that a higher dose ofB. coagulans(6.50mg/kg) is more effective in ameliorating the PCOS symptoms.