Search Results (18)

Extracellular synthesis of functional cyclodextrins (CDs) as intermediates of starch assimilation is a convenient microbial adaptation to sequester substrates, increase the half-life of the carbon source, carry bioactive compounds, and alleviate chemical toxicity through the formation of CD-guest complexes. Bacteria encoding the four steps of the carbohydrate metabolism pathway via cyclodextrins (CM-CD) actively internalize CDs across the microbial membrane via a putative type I ATP-dependent ABC sugar importer system, MdxEFG-(X/MsmX). While the first step of the CM-CD pathway encompasses extracellular starch-active cyclomaltodextrin glucanotransferases (CGTases) to synthesize linear dextrins and CDs, it is the ABC importer system in the second step that is the critical factor in determining which molecules from the CGTase activity will be internalized by the cell. Here, structure-function relationship studies of the cyclo⁄maltodextrin-binding protein MdxE of the MdxEFG-MsmX importer system from Thermoanaerobacter mathranii subsp. mathranii A3 are presented. Calorimetric and fluorescence studies of recombinant MdxE using linear dextrins and CDs showed that although MdxE binds linear dextrins and CDs with high affinity, the open-to-closed conformational change is solely observed after α- and β-CD binding, suggesting that the CM-CD pathway from Thermoanaerobacterales is exclusive for cellular internalization of these molecules. Structural analysis of MdxE coupled with docking simulations showed an overall architecture typically found in sugar-binding proteins (SBPs) that comprised two N- and C-domains linked by three small hinge regions, including the conserved aromatic triad Tyr193/Trp269/Trp378 in the C-domain and Phe87 in the N-domain involved in CD recognition and stabilization. Structural bioinformatic analysis of the entire MdxFG-MsmX importer system provided further insights into the binding, internalization, and delivery mechanisms of CDs. Hence, while the MdxE-CD complex couples to the permease subunits MdxFG to deliver the CD into the transmembrane channel, the dimerization of the cytoplasmatic promiscuous ATPase MsmX triggers active transport into the cytoplasm. This research provides the first results on a novel thermofunctional SBP and its role in the internalization of CDs in extremely thermophilic bacteria. Full article

Baicalin is a biologically active flavone glucuronide with poor water solubility that can be enhanced via glucosylation. In this study, the transglucosylation of baicalin was successfully achieved with CGTases from Thermoanaerobacter sp. and Bacillus macerans using α-cyclodextrin as a glucosyl donor. The synthesis of baicalin glucosides was optimized with CGTase from Thermoanaerobacter sp. Enzymatically modified baicalin derivatives were α-glucosylated with 1 to 17 glucose moieties. The two main glucosides were identified as Baicalein-7-O-α-D-Glucuronidyl-(1→4′)-O-α-D-Glucopyranoside (BG₁) and Baicalein-7-O-α-D-Glucuronidyl-(1→4′)-O-α-D-Maltoside (BG₂), thereby confirming recent findings reporting that glucuronyl groups are acceptors of this CGTase. Optimized conditions allowed for the attainment of yields above 85% (with a total glucoside content higher than 30 mM). BG₁ and BG₂ were purified via centrifugal partition chromatography after an enrichment through deglucosylation with amyloglucosidase. Transglucosylation increased the water solubility of BG₁ by a factor of 188 in comparison to that of baicalin (molar concentrations), while the same value for BG₂ was increased by a factor of 320. Finally, BG₁ and BG₂ were evaluated using antioxidant and anti-glycation assays. Both glucosides presented antioxidant and anti-glycation properties in the same order of magnitude as that of baicalin, thereby indicating their potential biological activity. Full article

The enzymatic transglycosylation of steviol glycosides can improve the edulcorant quality of steviol glycosides. Cyclodextrin glucanotransferase (CGTase) is one of the most popular glucanotransferases applied in this reaction. Herein, the CGTase-producing strain Alkalihalobacillus oshimensis CGMCC 23164 was isolated from Stevia planting soil. Using mass spectrometry-based secretome profiling, a high-efficiency CGTase that converted steviol glycosides to glucosylated steviol glycosides was identified and termed CGTase-13. CGTase-13 demonstrated optimal transglycosylation activity with 10 g/L steviol glycoside and 50 g/L soluble starch as substrates at <40 °C. Under the above conditions, the conversion rate of stevioside and rebaudioside A, two main components of steviol glycosides, reached 86.1% and 90.8%, respectively. To the best of our knowledge, this is the highest conversion rate reported to date. Compared with Toruzyme^® 3.0 L, the commonly used commercial enzyme blends, glucosylated steviol glycosides produced using CGTase-13 exhibited weaker astringency and unpleasant taste, faster sweetness onset, and stronger sweetness intensity. Thus, CGTase provides a novel option for producing high-quality glucosylated steviol glycoside products and has great potential for industrial applications. Full article

Amylomaltase is a well-known glucan transferase that can produce large ring cyclodextrins (LR-CDs) or so-called cycloamyloses via cyclization reaction. Amylomaltases have been found in several microorganisms and their optimum temperatures are generally around 60–70 °C for thermostable amylomaltases and 30–45 °C for the enzymes from mesophilic bacteria and plants. The optimum pHs for mesophilic amylomaltases are around pH 6.0–7.0, while the thermostable amylomaltases are generally active at more acidic conditions. Size of LR-CDs depends on the source of amylomaltases and the reaction conditions including pH, temperature, incubation time, and substrate. For example, in the case of amylomaltase from Corynebacterium glutamicum, LR-CD productions at alkaline pH or at a long incubation time favored products with a low degree of polymerization. In this review, we explore the synthesis of LR-CDs by amylomaltases, structural information of amylomaltases, as well as current applications of LR-CDs and amylomaltases. Full article

The use of unmodified starch in frozen foods can cause extremely undesirable textural changes after the freeze-thaw process. In this study, using cyclodextrin glucanotransferase (CGTase) and branching enzymes, an amylopectin cluster with high freeze-thaw stability was produced, and was named CBAC. It was found to have a water solubility seven times higher, and a molecular weight 77 times lower, than corn starch. According to the results of a differential scanning calorimetry (DSC) analysis, dough containing 5% CBAC lost 19% less water than a control dough after three freeze-thaw cycles. During storage for 7 days at 4 °C, bread produced using CBAC-treated dough exhibited a 14% smaller retrogradation peak and 37% less hardness than a control dough, suggesting that CBAC could be a potential candidate for clean label starch, providing high-level food stability under repeated freeze-thaw conditions. Full article

Amylomaltases (4-α-glucanotransferases, E.C. 2.4.1.25) are enzymes which can perform a double-step catalytic process, resulting in a transglycosylation reaction. They hydrolyse glucosidic bonds of α-1,4′-d-glucans and transfer the glucan portion with the newly available anomeric carbon to the 4′-position of an α-1,4′-d-glucan acceptor. The intramolecular reaction produces a cyclic α-1,4′-glucan. Amylomaltases can be found only in prokaryotes, where they are involved in glycogen degradation and maltose metabolism. These enzymes are being studied for possible biotechnological applications, such as the production of (i) sugar substitutes; (ii) cycloamyloses (molecules larger than cyclodextrins), which could potentially be useful as carriers and encapsulating agents for hydrophobic molecules and also as effective protein chaperons; and (iii) thermoreversible starch gels, which could be used as non-animal gelatin substitutes. Extremophilic prokaryotes have been investigated for the identification of amylomaltases to be used in the starch modifying processes, which require high temperatures or extreme conditions. The aim of this article is to present an updated overview of studies on amylomaltases from extremophilic Bacteria and Archaea, including data about their distribution, activity, potential industrial application and structure. Full article

Ganoderma lucidum is a medicinal fungus whose numerous triterpenoids are its main bioactive constituents. Although hundreds of Ganoderma triterpenoids have been identified, Ganoderma triterpenoid glycosides, also named triterpenoid saponins, have been rarely found. Ganoderic acid A (GAA), a major Ganoderma triterpenoid, was synthetically cascaded to form GAA-15-O-β-glucopyranoside (GAA-15-G) by glycosyltransferase (BtGT_16345) from Bacillus thuringiensis GA A07 and subsequently biotransformed into a series of GAA glucosides by cyclodextrin glucanotransferase (Toruzyme^® 3.0 L) from Thermoanaerobacter sp. The optimal reaction conditions for the second-step biotransformation of GAA-15-G were found to be 20% of maltose; pH 5; 60 °C. A series of GAA glucosides (GAA-G2, GAA-G3, and GAA-G4) could be purified with preparative high-performance liquid chromatography (HPLC) and identified by mass and nucleic magnetic resonance (NMR) spectral analysis. The major product, GAA-15-O-[α-glucopyranosyl-(1→4)-β-glucopyranoside] (GAA-G2), showed over 4554-fold higher aqueous solubility than GAA. The present study demonstrated that multiple Ganoderma triterpenoid saponins could be produced by sequential actions of BtGT_16345 and Toruzyme^®, and the synthetic strategy that we proposed might be applied to many other Ganoderma triterpenoids to produce numerous novel Ganoderma triterpenoid saponins in the future. Full article

While testing the ability of cyclodextrin glucanotransferases (CGTases) to glucosylate a series of flavonoids in the presence of organic cosolvents, we found out that this enzyme was able to glycosylate a tertiary alcohol (tert-butyl alcohol). In particular, CGTases from Thermoanaerobacter sp. and Thermoanaerobacterium thermosulfurigenes EM1 gave rise to the appearance of at least two glycosylation products, which were characterized by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as tert-butyl-α-D-glucoside (major product) and tert-butyl-α-D-maltoside (minor product). Using partially hydrolyzed starch as glucose donor, the yield of transglucosylation was approximately 44% (13 g/L of tert-butyl-α-D-glucoside and 4 g/L of tert-butyl-α-D-maltoside). The synthesized tert-butyl-α-D-glucoside exhibited the typical surfactant behavior (critical micellar concentration, 4.0–4.5 mM) and its properties compared well with those of the related octyl-α-D-glucoside. To the best of our knowledge, this is the first description of an enzymatic α-glucosylation of a tertiary alcohol. Full article

Cyclodextrin glucanotransferases (CGTases) convert starch to cyclodextrins (CD) of various sizes. To engineer a CGTase for the synthesis of large-ring CD composed of 9 to 12 glucose units, a loop structure of the protein involved in substrate binding was targeted for semi-rational mutagenesis. Based on multiple protein alignments and protein structure information, a mutagenic megaprimer was designed to encode a partial randomization of eight amino acid residues within the loop region. The library obtained encoding amino acid sequences occurring in wild type CGTases in combination with a screening procedure yielded sequences displaying a changed CD product specificity. As a result, variants of the CGTase from the alkaliphilic Bacillus sp. G825-6 synthesizing mainly CD9 to CD12 could be obtained. When the mutagenesis experiment was performed with the CGTase G825-6 variant Y183R, the same loop alterations that increased the total CD synthesis activity resulted in lower activities of the variant enzymes created. In the presence of the amino acid residue R183, the synthesis of CD8 was suppressed and larger CD were obtained as the main products. The alterations not only affected the product specificity, but also influenced the thermal stability of some of the CGTase variants indicating the importance of the loop structure for the stability of the CGTase. Full article

Ginsenosides from Panax ginseng (Korean ginseng) are unique triterpenoidal saponins that are considered to be responsible for most of the pharmacological activities of P. ginseng. However, the various linkage positions cause different pharmacological activities. In this context, we aimed to synthesize new derivatives of ginsenosides with unusual linkages that show enhanced pharmacological activities. Novel α-glycosylated derivatives of ginsenoside F1 were synthesized from transglycosylation reactions of dextrin (sugar donor) and ginsenoside F1 (acceptor) by the successive actions of Toruzyme^®3.0L, a cyclodextrin glucanotransferase. One of the resultant products was isolated and identified as (20S)-3β,6α,12β-trihydroxydammar-24ene-(20-O-β-D-glucopyranosyl-(1→2)-α-D-glucopyranoside) by various spectroscopic characterization techniques of fast atom bombardment-mass spectrometry (FAB-MS), infrared spectroscopy (IR), proton-nuclear magnetic resonance (¹H-NMR), ¹³C-NMR, gradient heteronuclear single quantum coherence (gHSQC), and gradient heteronuclear multiple bond coherence (gHMBC). As expected, the novel α-glycosylated ginsenoside F1 (G1-F1) exhibited increased solubility, lower cytotoxicity toward human dermal fibroblast cells (HDF), and higher tyrosinase activity and ultraviolet A (UVA)-induced inhibitory activity against matrix metalloproteinase-1 (MMP-1) than ginsenoside F1. Since F1 has been reported as an antiaging and antioxidant agent, the enhanced efficacies of the novel α-glycosylated ginsenoside F1 suggest that it might be useful in cosmetic applications after screening. Full article

The regioselective α-glucosylation of hesperetin was achieved by a transglycosylation reaction catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. using soluble starch as glucosyl donor. By combining mass spectrometry (ESI-TOF) and 2D-NMR analysis, the main monoglucosylated derivative was fully characterized (hesperetin 7-O-α-d-glucopyranoside). In order to increase the yield of monoglucoside, several reaction parameters were optimized: Nature and percentage of cosolvent, composition of the aqueous phase, glucosyl donor, temperature, and the concentrations of hesperetin and soluble starch. Under the optimal conditions, which included the presence of 30% of bis(2-methoxyethyl) ether as cosolvent, the maximum concentration of monoglucoside was approximately 2 mM, obtained after 24 h of reaction. To our knowledge, this is the first report of direct glucosylation of hesperetin employing free enzymes instead of whole cells. Full article

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙⁺ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages. Full article

Mogroside IIE is a bitter triterpenoid saponin which is the main component of unripe Luo Han Guo fruit and a precursor of the commercially available sweetener mogroside V. In this study, we developed an enzymatic glycosyl transfer method, by which bitter mogroside IIE could be converted into a sweet triterpenoid saponin mixture. The reactant concentration, temperature, pH and buffer system were studied. New saponins with the α-glucose group were isolated from the resulting mixtures, and the structures of three components of the extract were determined. The structure-taste relationships of these derivatives were also studied together with those of the natural mogrosides. The number and stereoconfiguration of glucose groups present in the mogroside molecules were found to be the main factor to determine the sweet or bitter taste of a compound. The antioxidant and food safety properties were initially evaluated by their radical scavenging ability and via 7 day mice survival tests, respectively. The results showed that the sweet triterpenoid saponin mixture has the same favorable physiological and safety characteristics as the natural mogrosides. Full article

Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as “parent CGTase” herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected. Full article

Rutin was subjected to intermolecular transglycosylation assisted with microwave irradiation using cyclodextrin glucanotransferase (CGTase) produced from Bacillus sp. SK13.002. Compared with the conventional enzymatic method for rutin transglycosylation (without microwave irradiation), microwave-assisted reaction (MAR) was much faster and thus more efficient. While the conventional reaction took dozens of hours to reach the highest conversion rate of rutin and yield of transglycosylated rutin, MAR of rutin transglycosylation completed within only 6 min providing almost the same conversion rate of rutin and yield of products consisting of mono-, di-, tri-, tetra-, penta-glucosylated rutins. The optimum transglycosylation conditions for microwave irradiation were 40 °C and 60 W with the reaction system consisting mainly of the mixture of 0.3 g rutin (0.49 mmol) pre-dissolved in 15 mL methanol, 1.8 g maltodextrin in 15 mL of 0.2 M sodium acetate buffer (pH 5.5) and CGTase (900 U). Results from this study indicated that MAR could be a potentially useful and economical technique for a faster and more efficient transglycosylation of rutin. Full article