Search Results (14)

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. Full article

This study aimed to synthesize maltitol using recombinant CGTase from Bacillus circulans A11 with β-cyclodextrin (β-CD) and sorbitol as a glucosyl donor and acceptor, respectively, and assess its antibacterial activity. Optimal conditions for producing the highest yield, 25.0% (w/w), were incubation of 1% (w/v) β-CD and sorbitol with 400 U/mL of CGTase in 20 mM phosphate buffer at pH 6.0 and 50 °C for 72 h. Subsequently, maltitol underwent large-scale production and was purified by HPLC. By mass spectrometry, the molecular weight of the synthesized maltitol was 379.08 daltons, corresponding exactly to that of standard maltitol. The relative sweetness of synthesized and standard maltitol was ~90% of that of sucrose. Spot assay on the agar plate showed that maltitol inhibited the growth of Streptococcus mutans DMST 18777 cells. In addition, the MIC and MBC values of synthesized and standard maltitol against S. mutans were also determined as 20 and 40 mg/mL, respectively. These results show that the synthesized maltitol can be produced at high yields and has the potential to be used as an anticariogenic agent in products such as toothpaste. 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

This study was designed to investigate the structure of synthesized hesperidin glycosides (HGs) and evaluate their antibacterial and α-glucosidase inhibitory activities. The preliminary structure of HG_s was confirmed by glucoamylase treatment and analyzed on thin layer chromatography (TLC). The LC-MS/MS profiles of HGs showed the important fragments at m/z ratios of 345.21 (added glucose to glucose of rutinose in HG₁) and 687.28 (added maltose to glucose of rutinose in HG₂), confirming that the structures of HG₁ and HG₂ were α-glucosyl hesperidin and α-maltosyl hesperidin, respectively. In addition, ¹H and ¹³C-NMR of hesperidin derivatives were performed to identify their α-1,4-glycosidic bonds. The MIC and MBC studies showed that transglycosylated HG₁ and HG₂ had better antibacterial and bactericidal activities than hesperidin and diosmin, and were more active against Staphylococcus aureus than Escherichia coli. Hesperidin, HG₁, HG₂, and diosmin inhibited α-glucosidase with IC₅₀ values of 2.75 ± 1.57, 2.48 ± 1.61, 2.36 ± 1.48, and 2.99 ± 1.23 mg/mL, respectively. The inhibition kinetics of HG₂ shown by a Lineweaver–Burk plot confirmed HG₂ was an α-glucosidase competitive inhibitor with an inhibitor constant, K_i, of 2.20 ± 0.10 mM. Thus, HGs have the potential to be developed into antibacterial drugs and treatments for treating α-glucosidase-associated type 2 diabetes. Full article

Cyclodextrins (CDs) and their derivatives have attracted significant attention in the pharmaceutical, food, and textile industries, which has led to an increased demand for their production. CD is typically produced by the action of cyclodextrin glycosyltransferase (CGTase) on starch. Owing to the relatively high cost of enzymes, the economic feasibility of the entire process strongly depends on the effective retention and recycling of CGTase in the reaction system, while maintaining its stability. CGTase enzymes immobilized on various supports such as porous glass beads or glyoxyl-agarose have been previously used to achieve this objective. Nevertheless, the attachment of biocatalysts on conventional supports is associated with numerous drawbacks, including enzyme leaching prominent in physical adsorption, reduced activity as a result of chemisorption, and increased mass transfer limitations. Recent reports on the successful utilization of metal–organic frameworks (MOFs) as supports for various enzymes suggest that CGTase could be immobilized for enhanced production of CDs. The three-dimensional microenvironment of MOFs could maintain the stability of CGTase while posing minimal diffusional limitations. Moreover, the presence of different functional groups on the surfaces of MOFs could provide multiple points for attachment of CGTase, thereby reducing enzyme loss through leaching. The present review focuses on the advantages MOFs can offer as support for CGTase immobilization as well as their potential for application in CD production. Full article

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli. Full article

A novel cyclodextrin (CD)-based controlled release system was developed in the small intestine to control the rate of drug release, on the premise of enteric-coated tablets. The system was designed based on the enzymes exogenous β-cyclodextrin glycosyltransferase (β-CGTase) and endogenous maltase-glucoamylase (MG), wherein MG is secreted in the small intestine and substituted by a congenerous amyloglucosidase (AG). The vanillin-/curcumin-β-CD complexes were prepared and detected by Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC), and host CD degradation was measured based on the glucose yield. The combination of β-CGTase and AG was also functional in the CD complex system. The variations in the concentrations of added β-CGTase, with AG constantly in excess, could effectively alter the rate of host CD degradation and guest release by monitoring glucose production and color disappearance, thus, demonstrating that guest release in the CD complex system could be precisely controlled by changing the amount of β-CGTase used. Thus, the in vitro simulation of the system indicated that a novel controlled release system, based on endogenous MG, could be established in the small intestine. The CD-based controlled release system can be potentially applied in drug delivery and absorption in the small intestine. 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

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

Palm curtain was selected as carrier to immobilize Bacillus circulans ATCC 21783 to produce β-cyclodextrin (β-CD). The influence for immobilization to CGTase activity was analyzed to determine the operation stability. 83.5% cyclodextrin glycosyltransferases (CGTase) of the 1st cycle could be produced in the 7th cycle for immobilized cells, while only 28.90% CGTase was produced with free cells. When palm curtain immobilized cells were reused at the 2th cycle, enzyme activities were increased from 5003 to 5132 U/mL, which was mainly due to physical adsorption of cells on palm curtain with special concave surface structure. Furthermore, conditions for expanded culture of immobilized cells in a 5 L fermentation tank were optimized through specific rotation speed procedure (from 350 r/min to 450 r/min with step size of 50 r/min) and fixed ventilation capacity (4.5 L/min), relations between biomass, enzyme activity, pH, and oxygen dissolution was investigated, and the fermentation periods under the two conditions were both 4 h shorter. Compared with free cell, immobilized cell was more stable, effective, and had better application potential in industries. 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

Glycoside hydrolases (GH) are enzymes capable to hydrolyze the glycosidic bond between two carbohydrates or even between a carbohydrate and a non-carbohydrate moiety. Because of the increasing interest for industrial applications of these enzymes, the immobilization of GH has become an important development in order to improve its activity, stability, as well as the possibility of its reuse in batch reactions and in continuous processes. In this review, we focus on the broad aspects of immobilization of enzymes from the specific GH families. A brief introduction on methods of enzyme immobilization is presented, discussing some advantages and drawbacks of this technology. We then review the state of the art of enzyme immobilization of families GH1, GH13, and GH70, with special attention on the enzymes β-glucosidase, α-amylase, cyclodextrin glycosyltransferase, and dextransucrase. In each case, the immobilization protocols are evaluated considering their positive and negative aspects. Finally, the perspectives on new immobilization methods are briefly presented. Full article

Four new minor diterpene glycosides with a rare α-glucosyl linkage were isolated from a cyclodextrin glycosyltransferase glucosylated stevia extract containing more than 98% steviol glycosides. The new compounds were identified as 13-[(2-O-β-D-glucopyranosyl-3-O-(4-O-α-D-glucopyranosyl)-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-α-D-glucopyranosyl-β-D-glucopyranosyl) ester] (1), 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-(4-O-(4-O-α-D-glucopyranosyl)-α-D-glucopyranosyl)-α-D-glucopyranosyl)-β-D-glucopyranosyl ester] (2), 13-[(2-O-β-D-glucopyranosyl-3-O-(4-O-(4-O-(4-O-α-D-glucopyranosyl)-α-D-glucopyranosyl)-α-D-glucopyranosyl)-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester (3), and 13-[(2-O-β-D-glucopyranosyl-3-O-(4-O-(4-O-(4-O-α-D-glucopyranosyl)-α-D-glucopyranosyl)-α-D-glucopyranosyl)-β-D-glucopyranosyl- β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-α-D-glucopyranosyl-β-D-glucopyranosyl) ester] (4) on the basis of extensive NMR and mass spectral (MS) data as well as hydrolysis studies. Full article

Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg⁻¹ protein, 20.0 U mg⁻¹ protein and 11.0 U mg⁻¹ protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl₂. K_m and V_max values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co²⁺, Zn²⁺, Cu²⁺, Hg²⁺, Ba²⁺, Cd²⁺, and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry. Full article