Cycloamylose Research Articles

Improved low water solubility of fisetin by enzymatic encapsulation reaction using cycloamylose produced by cyclodextrin glucanotransferase

Fisetin was encapsulated with cycloamylose (CA), an intermediate product of cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19), which is known to produce cyclodextrin (CD). The molecular weight measurement of the cycloamylose-fisetin inclusion complex (CA-FST) confirmed that it contained fisetin and CA with a degree of polymerization (DP) of 9–18. The aqueous solubility of CA-FST was approximately 3700 times higher than that of fisetin, with a solubility of 37.1 mg/mL. CA-FST showed a stability of 97.9 % at 4 °C over 1 week. CA-FST dissolved in water exhibited about 80 % antioxidant activity compared to the same amount of fisetin dissolved in methanol. In the analysis of anti-inflammatory effects, CA-FST decreased cytokine expression more effectively than fisetin, indicating that CA encapsulation using CGTase could be an effective method for overcoming the limited solubility of fisetin.

Development of positively-charged cycloamylose, CAQ as efficient nanodelivery system for siRNA

Cycloamylose (CA) can entrap molecules to form inclusion complexes. Here, we report the potential application of enzymatically produced CA from low-cost tapioca starch as a nanocarrier for human papillomavirus type 16 (HPV16) E6 siRNA in cervical cancer treatment. The cationic charged CA (CAQ) was produced by the introduction of (3-Chloro-2 hydroxypropyl) trimethylammonium chloride (Q188) to the particle, resulting in successful complexation of CAQ and siRNA. This was then transfected into the cervical cancer cell line (CaSki). The CAQ-siRNA complex demonstrated no cytotoxicity in normal cells and exhibited the same level of transfection efficiency compared to a commercial liposome-based particle. In contrast, the commercial liposome-based particles showed a 40 % decrease in the viability of normal cells. These results suggest that low-cost, high biocompatibility and easy-to-use CAQ is a feasible alternative for an siRNA carrier. This new system had no impact on cell viability and has the potential to be used in medical applications.

Physicochemical characteristics and in vitro bioavailability of licorice (Glycyrrhiza glabra L.) extract complexed using cyclic glucans

Licorice extract (LE), whose main ingredient is glabridin (GL), have various biological properties such as antioxidant and anticancer activities, however, their low solubility and bioavailability limit their application in the pharmaceutical and food industries. In this study, inclusion complexes (ICs) were developed using β-cyclodextrin (CD), 2-hydroxypropyl-β-cyclodextrin (HP), and cycloamylose (CA) to overcome the application limits of LE. The IC with cyclic glucans successfully enhanced the phase solubility of LE, and the stoichiometry of the complexes was investigated. After in vitro digestion, the bioaccessibility (% retention) of LE increased significantly in the IC samples, particularly with CA, compared to free LE. In vitro bioavailability, which was assessed using the results of bioaccessibility via the simulated gastrointestinal tract (GIT) model and permeability via Caco-2 monolayer assay, increased 2.5, 2.3, and 4.7-fold in the CD, HP, and CA ICs, respectively. This study provides a promising strategy to overcome the application limits of LE in various industries.

Improving solubility and stability of fat-soluble vitamins (A, D, E, and K) using large-ring cycloamylose

Fat-soluble vitamins (FSV) have beneficial biological activities, but their application is limited due to low solubility and stability. We investigated the capability of cycloamylose (CA) to improve the solubility and stability of FSV by forming a complex. The PXRD, FTIR, fluorescence spectrometry, 1D NMR, and 2D NMR analyses supported the complex formation between CA and FSV in both solution and solid states. Especially, interaction between FSV hydrophobic moiety and CA interior was elucidated by NMR. The solubility of FSV continuously increased without aggregation in 5% ethanol solution at 30 °C with CA concentration of up to 60 mM, while it either reached to plateau or showed a maximum value with the concentration of cyclodextrin (CD) and maltodextrin (MD), commercial agents. As CA concentration increased, a photo-degradation rate of FSV significantly reduced in 5% ethanol solution at room temperature (RT). Highly soluble and stable characteristics of CA, especially at high concentration, were responsible for the enhanced solubility and stability of FSV upon complexation with CA, suggesting the potential usage of CA as a promising host molecule.

Retarding Oxidative and Enzymatic Degradation of Phenolic Compounds Using Large-Ring Cycloamylose.

The phenolic compounds (PCs) abundant in fruits and vegetables are easily browned by oxygen and browning enzymes, with subsequent destruction of nutrients during food processing and storage. Therefore, natural anti-browning additives are required to control these reactions. The aim of the present study was to investigate the feasibility of cycloamylose (CA) complexation as a way to improve stability of PCs against oxidation and browning enzymes. The complex was prepared by reacting enzymatically produced CA with a degree of polymerization of 23–45 with PCs in aqueous solution. No significant differences were observed between the PCs and their CA complexes in 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging experiments. However, the reduction rate of their antioxidant activity was clearly reduced in the presence of CA for as long as 4 weeks. At the studied concentrations, the activity of polyphenol oxidase on all of the tested PC species was inhibited in the presence of CA, although this effect was less evident as the substrate concentration increased. The higher the CA concentration added to apple juice, the lower the variation in the total color difference (ΔE*) during storage, confirming that CA could be used as an effective natural anti-browning agent. Our study is the first to study the potential of CA as a natural material for browning control. The results obtained will provide useful information for active food applications requiring oxidative stability in fruit products.

Development of an enzymatic encapsulation process for a cycloamylose inclusion complex with resveratrol

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cycloamyloses (CAs), which are large cyclic glucans, and subsequently transforms them to α-, β-, and γ-cyclodextrins. We developed a novel encapsulation process based on the cyclization activity of CGTase and applied it to the formation of CA inclusion complexes with resveratrol (RVT), which has limited bioavailability due to its low water solubility. The encapsulated RVT (CA-RVT) was purified using preparative high-performance liquid chromatography. The water solubility of CA-RVT was 6,000-fold higher than that of RVT. CA-RVT in water demonstrated 98% stability for 1 week at 4 °C. According to radical scavenging activity and anti-inflammatory assays, CA-RVT in aqueous solution exhibited similar activities as an equal amount of RVT in dimethyl sulfoxide, suggesting the limited solubility of RVT can be overcome through CA encapsulation by CGTase, thus enhancing its nutraceutical value as a functional ingredient in the food industry.

Comparison of Catalyzing Properties of Bacterial 4-α-Glucanotransferases Focusing on Their Cyclizing Activity.

A newly cloned 4-α-glucanotransferase (αGT) from Deinococcus geothermalis and two typical bacterial αGTs from Thermus scotoductus and Escherichia coli (MalQ) were investigated. Among 4 types of catalysis, the cyclization activity of αGTs that produces cycloamylose (CA), a valuable carbohydrate making inclusion complexes, was intensively studied. The new αGT, DgαGT, showed close protein sequence to the αGT from T. scotoductus (TsαGT). MalQ was clearly separated from the other two αGTs in the phylogenetic and the conserved regions analyses. The reaction velocities of disproportionation, cyclization, coupling, and hydrolysis of three αGTs were determined. Intriguingly, MalQ exhibited more than 100-fold lower cyclization activity than the others. To lesser extent, the disproportionation activity of MalQ was relatively low. DgαGT and TsαGT showed similar kinetics results, but TsαGT had nearly 10-fold lower hydrolysis activity than DgαGT. Due to the very low cyclizing activity of MalQ, DgαGT and TsαGT were selected for further analyses. When amylose was treated with DgαGT or TsαGT, CA with a broad DP range was generated immediately. The DP distribution of CA had a bimodal shape (DP 7 and 27 as peaks) for the both enzymes, but larger DPs of CA quickly decreased in the DgαGT. Cyclomaltopentaose, a rare cyclic sugar, was produced at early reaction stage and accumulated as the reactions went on in the both enzymes, but the increase was more profound in the TsαGT. Taken together, we clearly demonstrated the catalytic differences between αGT groups from thermophilic and pathogenic bacteria that and showed that αGTs play different roles depending on their lifestyle.

PH-dependent antioxidant stability of black rice anthocyanin complexed with cycloamylose

This study investigated the effects of cycloamylose (CA) as a complexing agent on the antioxidant activity and stability of anthocyanin extracted from black rice (BRA) at various pHs (2, 4, 6, and 8) and compared the results with those of β-cyclodextrin (βCD). The CA (DP 6–31) produced from Dodamssal rice starch had an anti-copigmentation effect for BRA and the discoloration effect increased with the complexing agent concentration, which supported the intramolecular interaction between them. With heat treatment, the CA-BRA complex had a greater antioxidant stability than did free BRA at pHs 4, 6, and 8 in a concentration-dependent manner. In particular, BRA complexed with 1% CA at pH 4 had an approximately 2.0-fold lower degradation rate (kd) than that of free BRA. This improvement in the thermal stability was comparable to or slightly better than that of 5% βCD. CA significantly improved the photostability of BRA at all pHs tested, similarly to βCD. CA might protect the moiety of BRA functional groups that are involved in antioxidant activity and susceptible to heat and light. The results demonstrated that CA could be an effective complexing agent for BRA and the stable CA-BRA complex could be used as a promising functional material for the various industries.

Size and shape of cycloamylose estimated using column chromatography coupled with small-angle X-ray scattering

We built a measurement system combining column chromatography and small angle X-ray scattering (SAXS) to efficiently investigate the dilute solution properties of a series of different molecules, and applied it to cycloamylose (CA), a large ring cyclodextrin. Two different column separation modes were used, size exclusion chromatography (SEC) and reverse phase chromatography with an octadecylsilane bonded silica (ODS) column. Both SEC- and ODS-SAXS worked well and the resulting data agreed with data obtained by batch SAXS experiments. Dilute solution properties of CA were studied for molecular weights ranging from 1621 (degree of polymerization DP = 10) to 9000 (DP > 55) using this flow cell-based SAXS system, and the radius of gyration increased monotonically with increasing DP. This means that individual CA molecules in water are flexible enough and do not adopt any characteristic folding structure at specific DPs, which is what the simulation literature suggested, which will be the basis of physicochemical properties of food polysaccharides as food modifiers.

Enhancing antioxidant and antimicrobial activity of carnosic acid in rosemary (Rosmarinus officinalis L.) extract by complexation with cyclic glucans

Of all the active compounds in rosemary extract, carnosic acid (CaA) has the most potent antimicrobial and antioxidant activity; however, its low solubility limits its applications. We developed complexing systems using cycloamylose (CA), branched dextrin (BD), and β-cyclodextrin (βCD) to improve the solubility of CaA and compared it to the use of maltodextrin (MD). The complexes formed with CA, BD, βCD, and MD improved the water solubility of CaA by as much as 2.8-fold, 2.1-fold, 1.75-fold, and 2.06-fold, respectively. The antioxidant capacity of CaA in aqueous solutions was also enhanced in the complexes due to the increased water solubility. Interestingly, the antimicrobial activity was improved more dramatically upon complexation with CA (7.27-fold) compared to the improvement when complexed with BD (4.82-fold), βCD (2.87-fold), and MD (3.83-fold). This may be due to the improvement of the antimicrobial potential of the functional groups of CaA by complexation with flexible cyclic glucans.

Physicochemical interactions of cycloamylose with phenolic compounds

The complex formation capability of cycloamylose (CA), having a degree of polymerization of 23–45, with phenolic compounds (PCs) was investigated using various physicochemical techniques. The fluorescence intensity of PCs increased and then reached a plateau at 10–20mM cyclodextrin, while it continued to increase at up to 60mM CA. Thermodynamic data of CA complexes with PCs revealed that the binding process was primarily enthalpy-driven and spontaneous. CA favored to form the most stable complex with chlorogenic acid (CHA) among all PCs. Chemical shift changes for the protons in interior and exterior of CA, as well as in PCs suggested a possible formation of both inclusion and extramolecular interactions between CA and PCs. The ROESY spectrum confirmed that the aromatic moieties of CHA were partially interacted with CA molecules through relatively weak binding. XRD, DSC, and SEM results also supported the complex formation by intermolecular interaction between CA and CHA.

High-yield cycloamylose production from sweet potato starch using Pseudomonas isoamylase and Thermus aquaticus 4-α-glucanotransferase.

An optimal reaction condition for producing cycloamylose (CA) from sweet potato starch was investigated using a combination of isoamylase (from Pseudomonas sp.) and 4-α-glucanotransferase (from Thermus aquaticus, TAαGT). Starch was debranched by isoamylase for 8 h and subsequently reacted with TAαGT for 12 h. The yield and purity of CA products were determined using HPSEC and MALDI-TOFMS, respectively. Consequently, the maximum yield was 48.56%, exhibiting the highest CA production efficiency ever reported from starch. The CA products showed a wide range of the degree of polymerization (DP) with the minimum DP of 5. CA was also produced by simultaneous treatment of isoamylase and TAαGT. The yield was 3.31%, and the final products were contaminated by multiple branched and linear molecules. This result suggests that a former reaction condition (the sequential addition of isoamylase and TAαGT) is preferable for producing CA from sweet potato starch.

Physicochemical properties of native and partially gelatinized high-amylose jackfruit (Artocarpus heterophyllus Lam.) seed starch

Jackfruit seed starch (JFSS) cultivated in Vietnam had a high amylose content (∼44%). Differential scanning calorimetry thermogram of JFSS consisted of two separate endothermic peaks with distinct onset and conclusion temperatures. When JFSS was heated at 70 °C, the first endothermic peak disappeared, whereas the second endotherm remained. The partially-gelatinized JFSS still possessed A-type X-ray diffraction pattern. SEM images showed that the rim of the starch granule at the hollow bottom of the bell shape first melted during the partial gelatinization at 70 °C. Both native and partially-gelatinized JFSS produced soft and highly elastic gels, but their moduli differed slightly. Additionally, the partially-gelatinized JFSS was successfully used to produce genistin–cycloamylose (CA) and amylose–lysolecithin complexes with and without 4-α-glucanotransferase. Water sorption isotherms of partially gelatinized JFSS showed higher water-holding capacity than that of the native starch.

Crystal structure of amylomaltase from Corynebacterium glutamicum

Amylomaltase (AM; EC 2.4.1.25) belongs to the 4-α-glucanotransferase (4αGTase) group of the α-amylase family. The enzyme can produce cycloamylose (CA) or large-ring cyclodextrin (LR-CD) through intramolecular transglycosylation or cyclization reactions of α-1,4 glucan. Amylomaltase from the mesophilic bacterium Corynebacterium glutamicum yielded different LR-CD production profile from that of the well-characterized Thermus aquaticus enzyme [1,2]. C. glutamicum amylomaltase (CgAM) was overexpressed, purified and crystallized [3]. X-ray crystal structure of CgAM differs from Th. aquaticus amylomaltase in the presence of an additional N-terminus domain. The acarbose- and maltotriose- bound structures revealed the residues involved in substrate binding.

Molecular mutagenesis at Tyr-101 of the amylomaltase transcribed from a gene isolated from soil DNA.

The wild-type (WT) amylomaltase gene was directly isolated from soil DNA and cloned into a pET19b vector to express in E. coli BL21(DE3). The ORF of this gene consisted of 1,572 bp, encoding an enzyme of 523 amino acids. Though showing 99% sequence identity to amylomaltse from Thermus thermophilus ATCC 33923, this enzyme is unique in its alkaline optimum pH. In order to alter amylomaltase with less coupling or hydrolytic activity to enhance cycloamylose (CA) formation through cyclization reaction, site-directed mutagenesis of the second glucan binding site involving in CA production was performed at Tyr-101. The result revealed that the mutated Y101S enzyme showed a small increase in cyclization activity while significantly decreased disproportionation, coupling and hydrolytic activities. Mutation also resulted in the change in substrate specificity for disproportionation reaction. The WT enzyme preferred maltotriose, while the activity of mutated enzyme was the highest with maltopentaose substrate. Product analysis by HPAEC-PAD demonstrated that the main CAs of the WT amylomaltase were CA29-CA37. Y101S mutation did not change the product pattern, however, the amount of CAs formed by the mutated enzyme tended to increase especially at long incubation time.

Cycloamylose production from amylomaize by isoamylase and Thermus aquaticus 4-α-glucanotransferase

Amylomaize (AMM) was utilized as the substrate for cycloamylose (CA) production in this study. After debranching, AMM was incubated with 10U/g of Thermus aquaticus 4-α-glucanotransferase (TA 4αGTase) for various reaction times in water or in DMSO reaction system. The maximum conversion yield of CA was greatly improved from 24.55% to 45.58% and from 27.40% to 47.25% after debranching in the water and DMSO reaction systems, respectively. Compared with the method that produced CA from commercial potato amylose, this method produced CA from a natural amylopectin containing starch with enhanced conversion yield after debranching. Meanwhile, we found that the minimum degree of polymerization (DP) of CA from TA 4αGTase treatment was 5, regardless of the reaction conditions. These results were different from those reported in the literature that stated the minimum DP of CA produced with a TA 4αGTase treatment was 22 regardless of the reaction conditions.

One-Pot Synthesis of Cycloamyloses from Sucrose by Dual Enzyme Treatment: Combined Reaction of Amylosucrase and 4-α-Glucanotransferase

Amylose-like α-(1,4)-glucan known as the most favorable substrate for the enzymatic production of cycloamyloses (CAs) using 4-α-glucanotransferase has a solubility issue, which requires an additional solubilization process. In our study, two glucosyltransferases, Synechocystis 4-α-glucanotransferase and Neisseria amylosucrase, were adopted to develop an efficient biocatalytic production process of CAs directly from sucrose. From one-pot synthesis, the maximum CA yield (9.6%, w/w) with 0.3 M sucrose was achieved with 10 units/mL of amylosucrase and 0.1 unit/mL of 4-α-glucanotransferase at 40 °C for a 3 h reaction in a simultaneous dual enzyme reaction mode. The size of linear α-(1,4)-glucan was positively related to the CA productivity by 4-α-glucanotransferase in a hyperbolic manner. Using our innovative bioprocess, there was no practical limitation on the initial sucrose concentration and no use of insoluble linear α-(1,4)-glucan substrate. Consequently, the concomitant dual enzyme reaction converted sucrose directly to CAs via in situ transient linear α-(1,4)-glucan as an soluble intermediate.

Isolation of cycloamylose by iodine affinity capillary electrophoresis

In contrast to α-, β- and γ-cyclodextrins, little information is available on the isolation and separation of cycloamylose (CA) with degree of polymerization (DP) larger than 22. The objective of the current study was to develop a new iodine affinity capillary electrophoresis (CE) for separation of CA with DP of 22–42, which was based on the formation of CA-iodine inclusion complexes, CA with twisted conformations made complicated mobility behaviors on CE instead of merely size dependent. The influences of iodide/iodine ratio, iodine concentration, pH, ion strength of running phosphate buffer, voltage, and temperature on the peak resolution and electrophoretic mobility were further investigated. Our results suggest that iodine affinity capillary electrophoresis provides a versatile and selective tool for the isolation and analysis of CA with DP from 22 to 42.

A novel amylomaltase from Corynebacterium glutamicum and analysis of the large-ring cyclodextrin products

Amylomaltase catalyzes the formation of large-ring cyclodextrins (LR-CDs) from starch. This study aims to construct the recombinant amylomaltase from Corynebacterium glutamicum and to characterize the purified enzyme with the emphasis on the profile of LR-CDs production. A novel amylomaltase from Corynebacterium glutamicum ATCC 13032 was cloned and expressed in Escherichia coli BL21 (DE3) using the expression vector pET-19b. The open reading frame of amylomaltase gene of 2,121 bp (encoding the polypeptide of 706 amino acid residues) was obtained with the N-terminal His-tag fragment of 69 bp attached before the start codon of the amylomaltase gene. The deduced amino acid sequence showed a low sequence identity (20–25%) to those thermostable amylomaltases from Thermus sp. The maximum enzyme activity was obtained when the recombinant cells were cultured at 37 °C for 2 h after induction with 0.4 mM isopropyl thio-β-D-galactoside (IPTG). The enzyme was 11-fold purified with a yield of 30% by a HiTrap affinity column. The purified amylomaltase showed a single band of 84 kDa on a 7.5% SDS-PAGE. When the enzyme acted on pea starch, it catalyzed an intramolecular transglucosylation (cyclization) reaction that produced LR-CDs or cycloamyloses (CA). The product profile was dependent on the incubation time and the enzyme concentration. Shorter incubation time gave larger LR-CDs as principal products. At 4 h incubation, the product was composed of a mixture of LR-CDs in the range of CD19–CD50, with CD27–28 as products with highest amount. It is noted that CD19 was the smallest product in all conditions tested. The enzyme also catalyzes intermolecular transglucosylation on various malto-oligosaccharides, with maltose as the smallest substrate.

Transglycosylation properties of maltodextrin glucosidase (MalZ) from Escherichia coli and its application for synthesis of a nigerose-containing oligosaccharide

The transglycosylation reaction of maltodextrin glucosidase (MalZ) cloned and purified from Escherichia coli K12 was characterized and applied to the synthesis of branched oligosaccharides. Purified MalZ preferentially catalyzed the hydrolysis of maltodextrin, γ-cyclodextrin (CD), and cycloamylose (CA). In addition, when the enzyme was incubated with 5% maltotriose (G3), a series of transfer products were produced. The resulting major transfer products, annotated as T1, T2, and T3, were purified and their structures were determined by TLC, MALDI-TOF/MS, 13C NMR, and enzymatic analysis. T1 was identified as a novel compound, maltosyl α-1,3-maltose, whereas T2 and T3 were determined to be isopanose and maltosyl-α-1,6-maltose, respectively. These results indicated that MalZ transferred sugar moiety mainly to C-3 or C-6–OH of glucose of the acceptor molecule. To obtain highly concentrated transfer products, the enzyme was reacted with 10% liquefied cornstarch, and then glucose and maltose were removed by immobilized yeast. The T1 content of the resulting reaction mixture reached 9.0%. The mixture of T1 containing a nigerose moiety can have an immunopotentiating effect on the human body and may be a potential functional sugar stuff.