Large-ring Cyclodextrin Research Articles

Research on the quantitative relationship of the viscosity reduction effect of large-ring cyclodextrin on potato starch during gelatinization process and mechanism analysis

Starch is extensively used across various fields due to its renewable properties and cost-effectiveness. Nonetheless, the high viscosity that arises from gelatinization poses challenges in the industrial usage of starch at high concentrations. Thus, it's crucial to explore techniques to lower the viscosity during gelatinization. In this study, large-ring cyclodextrins (LR-CDs) were synthesized from potato starch (PS) by using 4-α-glucanotransferase and then added to PS to alleviate the increased viscosity during gelatinization. The results from rapid viscosity analyzer (RVA) demonstrated that the inclusion of 5 % (w/w) LR-CDs markedly reduced the peak viscosity (PV) and final viscosity (FV) of PS by 49.85 % and 28.17 %. In addition, there was a quantitative relationship between PV and LR-CDs. The equation was fitted as y = 2530.73×e−x/2.48+1832.79, which provided a basis for the regulation of PS viscosity. The mechanism of LR-CDs reducing the viscosity of PS was also studied. The results showed that the addition of LR-CDs inhibited the gelatinization of PS by enhancing orderliness and limiting water absorption, resulting in a decrease in viscosity. This study provides a novel method for reducing the viscosity of starch, which is helpful for increasing its concentration and reducing energy consumption in industrial applications.

Effects of large-ring cyclodextrin produced by 4-α-glucosyltransferase on gelatinization and retrogradation behavior of starches

Gelatinization is an important step in starch processing to obtain viscosity, gelation, and other properties. However, both high viscosity during gelatinization and retrogradation during storage limit the processing efficiency and quality of starch products. Here, Large-ring cyclodextrin (LR-CD) was used to amend starch gelatinization and retrogradation behavior. Firstly, LR-CD was efficiently produced from potato starch (PS) using Thermoproteus uzoniensis 4-α-glucanotransferase, β-amylase, and pullulanase. The effects of LR-CD on the gelatinization, rheological properties, and retrogradation of PS, cassava starch (CAS), corn starch (CS), waxy corn starch (WCS), and pea starch (PEAS) were investigated. Rapid viscosity analyzer (RVA) results indicated that PS after the addition of LR-CD showed greatest decrease in viscosity, whereas viscosity of CAS, CS, WCS and PEAS was only little altered. The viscosity of PS thus decreased by 40.8% when 5% (w/w) LR-CD was added. Comparing granule sizes with RVA results suggested that the entry of LR-CD into the swelling starch granules during gelatinization was facilitated for larger starch granules, which thereby reduced the viscosity. Differential scanning calorimetry (DSC) analysis revealed the retrogradation enthalpy (ΔHr) of PS/LR-CD mixtures to decrease with increasing amounts of added LR-CD. LR-CD could inhibit retrogradation of the five starches by hindering starch molecular rearrangement. Moreover, the rheological tests revealed that there was an interaction between LR-CD and starch, resulting in less rigid starch gels. This study developed a novel method for adjusting starch behavior in food processing.

In Vitro and In Silico Study on the Molecular Encapsulation of α-Tocopherol in a Large-Ring Cyclodextrin.

α-tocopherol is the physiologically most active form of vitamin E, with numerous biological activities, such as significant antioxidant activity, anticancer capabilities, and anti-aging properties. However, its low water solubility has limited its potential use in the food, cosmetic, and pharmaceutical industries. One possible strategy for addressing this issue is the use of a supramolecular complex with large-ring cyclodextrins (LR-CDs). In this study, the phase solubility of the CD26/α-tocopherol complex was investigated to assess the possible ratios between host and guest in the solution phase. Next, the host-guest association of the CD26/α-tocopherol complex at different ratios of 1:2, 1:4, 1:6, 2:1, 4:1, and 6:1 was studied by all-atom molecular dynamics (MD) simulations. At 1:2 ratio, two α-tocopherol units interact spontaneously with CD26, forming an inclusion complex, as supported by the experimental data. In the 2:1 ratio, a single α-tocopherol unit was encapsulated by two CD26 molecules. In comparison, increasing the number of α-tocopherol or CD26 molecules above two led to self-aggregation and consequently limited the solubility of α-tocopherol. The computational and experimental results indicate that a 1:2 ratio could be the most suitable stoichiometry to use in the CD26/α-tocopherol complex to improve α-tocopherol solubility and stability in inclusion complex formation.

Identification of crucial amino acid residues involved in large ring cyclodextrin synthesis by amylomaltase from Corynebacterium glutamicum

Amylomaltase can be used to synthesize large ring cyclodextrins (LR-CDs), applied as drug solubilizer, gene delivery vehicle and protein aggregation suppressor. This study aims to determine the functional amino acid positions of Corynebacterium glutamicum amylomaltase (CgAM) involved in LR-CD synthesis by site-directed mutagenesis approach and molecular dynamic simulation. Mutants named Δ167, Y23A, P228Y, E231Y, A413F and G417F were constructed, purified, and characterized. The truncated CgAM, Δ167 exhibited no starch transglycosylation activity, indicating that the N-terminal domain of CgAM is necessary for enzyme activity. The P228Y, A413F and G417F produced larger LR-CDs from CD36-CD40 as compared to CD29 by WT. A413F and G417F mutants produced significantly low LR-CD yield compared to the WT. The A413F mutation affected all tested enzyme activities (starch tranglycosylation, disproportionation and cyclization), while the G417F mutation hindered the cyclization activity. P228Y mutation significantly lowered the kcat of disproportionation activity, while E231Y mutant exhibited much higher kcat and Km values for starch transglycosylation, compared to that of the WT. In addition, Y23A mutation affected the kinetic parameters of starch transglycosylation and cyclization. Molecular dynamic simulation further confirmed these mutations’ impacts on the CgAM and LR-CD interactions. Identified functional amino acids for LR-CD synthesis may serve as a model for future modification to improve the properties and yield of LR-CDs.

Formation and molecular dynamics simulation of inclusion complex of large-ring cyclodextrin and 4-terpineol.

In the present study, the formation and structure of the inclusion compound of large-ring cyclodextrin and 4-terpineol were obtained through different experiments and molecular dynamics (MD) simulation. The analysis of FTIR, 1 H-NMR, and thermodynamic results confirmed the formation of clathrates. Analysis of molecular structure (root-mean-square deviation and radius of gyration), solubility, and interaction energy (Coul, H bond) based on MD simulations further clarified the nature of the clathrate and the conformational changes caused by guest molecules as well as inclusion complexes process trends. The inclusion complex reportedly has a new crystal structure with improved thermal stability. PRACTICAL APPLICATION: This is the first work to demonstrate the complex formation between 4-terpineol and large-ring cyclodextrin by molecular dynamics simulation. Molecular dynamics simulation confirmed the formation of inclusion complexes theoretically. Conformational changes of the molecules and the formation of complexes with improved thermal stability were observed. Complexing with large-ring cyclodextrin can be used as an effective means to encapsulate the aroma/flavor compounds.

Enhancement of large ring cyclodextrin production using pretreated starch by glycogen debranching enzyme from Corynebacterium glutamicum

Synthesis of large-ring cyclodextrins (LR-CDs) in any significant amount has been challenging. This study enhanced the LR-CDs production by Thermus filiformis amylomaltase (TfAM) enzyme by starch pretreatment using glycogen debranching enzyme from Corynebacterium glutamicum (CgGDE). CgGDE pretreated tapioca starch gave LR-CD conversion of 31.2 ± 2.2%, compared with LR-CDs produced from non-treated tapioca starch (16.0 ± 2.4%). CgGDE pretreatment enhanced amylose content by approximately 30%. Notably, a shorter incubation time of 1 h is sufficient for CgGDE starch pretreatment to produce high LR-CD yield, compared with 6 h required for the commercial isoamylase. High-Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD) and Gel Permeable Chromatography (GPC) revealed that CgGDE is more efficient than the commercial isoamylase in debranching tapioca starch and gave lower molecular weight products. In addition, lower amount of by-products (linear oligosaccharides) were detected in cyclization reaction when using CgGDE-pretreated starch. In conclusion, CgGDE is a highly effective enzyme to promote LR-CD synthesis from starch with a shorter incubation time than the commercial isoamylase.

Solubility enhancement of poorly water soluble domperidone by complexation with the large ring cyclodextrin

The water solubility of domperidone (DMP) could be improved by complexation with large ring cyclodextrins (LR-CDs). LR-CDs contain a relatively hydrophobic cavity that is capable of entrapping the molecules to form inclusion complexes. The complex formation capability of mixture LR-CDs having a degree of polymerization (DP) of 22–48, with DMP was investigated. The phase solubility profile of mixture LR-CD/DMP was classified as AN-type, resulting in increased DMP solubility in water by 3-fold. Various physicochemical techniques confirmed the mixture LR-CD/DMP complex formation. Single LR-CD with DP of 26, 27, 28, 29, 30, 33 and 34 (CD26 ~ CD34) were isolated from LR-CD mixtures using ODS column for HPLC separation. The CD33/DMP complex has demonstrated the most significant improvement compared to other single LR-CD complexes with a 2.7-fold increase in DMP solubility. The molecular dynamic result revealed that DMP formed stable complexes with CD33 by positioned fully encapsulated inside the cavity and covered by 13–14 subunits of CD33.

Encapsulation of α-tocopherol in large-ring cyclodextrin containing 26 α-D-glucopyranose units: A molecular dynamics study

α-Tocopherol is the most biologically active form of vitamin E (VE) exhibiting various biological activities such as strong antioxidant activity, antitumor properties and antiaging effects. However, the low water solubility of α-tocopherol has limited its prospective use in food, cosmetic and pharmaceutical industry. One of the promising strategies to tackle such issue is the use of a supramolecular complex with cyclodextrins (CDs). In this study, the encapsulation of α-tocopherol with six different initial un-complexed states into the large-ring CD containing 26 α-D-glucopyranose units (CD26) was studied by means of 400-ns molecular dynamics (MD) simulations to investigate their host–guest association process and the preferential binding efficiency upon complexation at low concentration of α-tocopherol. The MD results show that α-tocopherol spontaneously interacted with CD26 within 20 ns and formed a stable inclusion complex during 150–400 ns. From all six independent simulations, the inclusion complexes can be divided into two groups, which were fully and partly encapsulated inside the cavity of CD26. The α-tocopherol of the first group was covered by 13–14 subunits of CD26, while α-tocopherol in another group was attached by 6–10 subunits of CD26. In addition, α-tocopherol was surrounded by one- or two-turn helix of one half-ring of CD26, leaving the rest of cavities for forming complexation with a larger number of α-tocopherol molecules. Therefore, CD26 can be used as a solubility and stability enhancer for α-tocopherol through inclusion complexation.

Streptococcus agalactiae amylomaltase offers insight into the transglycosylation mechanism and the molecular basis of thermostability among amylomaltases

Amylomaltase (AM) catalyzes transglycosylation of starch to form linear or cyclic oligosaccharides with potential applications in biotechnology and industry. In the present work, a novel AM from the mesophilic bacterium Streptococcus agalactiae (SaAM), with 18–49% sequence identity to previously reported AMs, was characterized. Cyclization and disproportionation activities were observed with the optimum temperature of 30 °C and 40 °C, respectively. Structural determination of SaAM, the first crystal structure of small AMs from the mesophiles, revealed a glycosyl-enzyme intermediate derived from acarbose and a second acarbose molecule attacking the intermediate. This pre-transglycosylation conformation has never been before observed in AMs. Structural analysis suggests that thermostability in AMs might be mainly caused by an increase in salt bridges since SaAM has a lower number of salt bridges compared with AMs from the thermophiles. Increase in thermostability by mutation was performed. C446 was substituted with A/S/P. C446A showed higher activities and higher kcat/Km values for starch in comparison to the WT enzyme. C446S exhibited a 5 °C increase in optimum temperature and the threefold increase in half-life time at 45 °C, most likely resulting from H-bonding interactions. For all enzymes, the main large-ring cyclodextrin (LR-CD) products were CD24-CD26 with CD22 as the smallest. C446S produced more CD35-CD42, especially at a longer incubation time.

Amylomaltase from Thermus filiformis: expression in Saccharomyces cerevisiae and its use in starch modification.

To express amylomaltase from Thermus filiformis (TfAM) in a generally recognized as safe (GRAS) organism and to use the enzyme in starch modification. TfAM was expressed in Saccharomyces cerevisiae, using 2% (w/v) galactose inducer under GAL1 promoter. The enzyme was thermostable with high disproportionation and cyclization activities. The main large-ring cyclodextrin (CD) products were CD24-CD29, with CD26 as maximum at all incubation times. TfAM was used to modify cassava and pea starches, the amylose content decreased 18% and 30%, respectively, when 5% (w/v) starch was treated with 0·5UTfAMg-1 starch. The increase in short branched chain (DP, degree of polymerization, 1-5) and the broader chain length distribution pattern which extended to the longer chain (DP40) after TfAM treatment were observed. The thermal property was changed, with an increase in retrogradation of starch as suggested by a lower enthalpy. TfAM was successfully expressed in S. cerevisiae and was used to make starches with new functionality. This is the first report on the expression of AM in the GRAS yeast and the production of a modified starch gel from pea starch to improve the versatility of starch for food use.

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.

Significance of H461 at subsite +1 in substrate binding and transglucosylation activity of amylomaltase from Corynebacterium glutamicum

Amylomaltase (AM) catalyzes inter- and intra-molecular transglycosylation reactions of glucan to yield linear and cyclic oligosaccharide products. The functional roles of the conserved histidine at position 461 in the active site of AM from Corynebacterium glutamicum (CgAM) was investigated. H461 A/S/D/R/W were constructed, their catalytic properties were compared to the wild-type (WT). A significant decrease in transglucosylation activities was observed, especially in H461A mutant, while hydrolysis activity was barely affected. The transglucosylation factor of the H461A-CgAM was decreased by 8.6 folds. WT preferred maltotriose (G3) as substrate for disproportionation reaction, but all H461 mutants showed higher preference for maltose (G2). Using G3 substrate, kcat/Km values of H461 mutated CgAMs were 40–64 folds lower, while the Km values were twice higher than those of WT. All mutants could not produce large-ring cyclodextrin (LR-CD) product. The heat capacity profile indicated that WT had higher thermal stability than H461A. The X-ray structure of WT showed two H-bonds between H461 and heptasaccharide analog at subsite +1, while no such bonding was observed from the model structure of H461A. The importance of H461 on substrate binding with CgAM was evidenced. We are the first to mutate an active site histidine in AM to explore its function.

Y418 in 410s loop is required for high transglucosylation activity and large-ring cyclodextrin production of amylomaltase from Corynebacterium glutamicum

Amylomaltase catalyzes α-1,4 glucosyl transfer reaction to yield linear or cyclic oligosaccharide products. The aim of this work is to investigate functional roles of 410s loop unique to amylomaltase from Corynebacterium glutamicum (CgAM). Site-directed mutagenesis of Y418, the residue at the loop tip, was performed. Y418A/S/D/R/W/F - CgAMs were characterized and compared to the wild-type (WT). A significant decrease in starch transglucosylation, disproportionation and cyclization activities was observed. Specificity for G3 substrate in disproportionation reaction was not changed; however, Y418F showed an increase in preference for longer oligosaccharides G5 to G7. The catalytic efficiency of Y418 mutated CgAMs, except for Y418F, was significantly lower (up to 8- and 12- fold for the W and R mutants, respectively) than that of WT. The change was in the kcat, not the Km values which were around 16–20 mM. The profile of large-ring cyclodextrin (LR-CD) product was different; the principal product of Y418A/D/S was shifted to the larger size (CD36-CD40) while that of the WT and Y418F peaked at CD29-CD33. The product yield was reduced especially in W and R mutants. Hence Y418 in 410s loop of CgAM not only contributes to transglucosylation activities but also controls the amount and size of LR-CD products through the proposed hydrophobic stacking interaction and the suitable distance of loop channel for substrate entering. This is the first report to show the effect of the loop tip residue on LR-CD product formation.

Roles of N287 in catalysis and product formation of amylomaltase from Corynebacterium glutamicum

Amylomaltase catalyzes intermolecular and intramolecular transglucosylation reactions to form linear and cyclic oligosaccharides, respectively. The aim of this work is to investigate the structure-function relationship of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM). Site-directed mutagenesis was performed to substitute Tyr for Asn287 (N287Y) to determine its role in controlling amylomaltase activity and product formation. Expression of the wild-type (WT) and N287Y was achieved by cultivating recombinant cells in the medium containing lactose at 16 °C for 14 h. The purified mutated enzyme showed a significant decrease in all transglucosylation activities while hydrolysis activity was not changed. Optimum temperature and pH for disproportionation reaction were slightly changed upon mutation while those for cyclization reaction were not changed. Interestingly, N287Y showed a change in large-ring cyclodextrin (LR-CD) product profile in which the larger size was observed together with an increase in thermostability and substrate preference for G5 in addition to G3. The secondary structure of the mutated enzyme was slightly changed in related to the WT as evidenced from circular dichroism analysis. This work thus demonstrates that N287 is required for transglucosylation activities of CgAM. Having an aromatic residue in this position increased thermostability, changed product profile and substrate preference but demolished most enzyme activities.

Optimization of large-ring cyclodextrin production from starch by amylomaltase from Corynebacterium glutamicum and effect of organic solvent on product size.

To increase yield of starch conversion to large-ring cyclodextrins (LR-CDs) by amylomaltase from Corynebacterium glutamicum (CgAM). In this work, LR-CDs produced from pea, tapioca, corn, potato, rice and glutinous-rice starch by the recombinant CgAM were analysed by High-Performance Anion-Exchange Chromatography Using Pulsed Amperometric Detection (HPAEC-PAD). Among these, pea starch gave the highest yield of LR-CDs. Pretreatment of pea starch with isoamylase prior to incubation with CgAM resulted in the increase in LR-CD products by 20%. Surprisingly, CgAM converted starch into LR-CDs within a wide pH range (pH 5·5-9·0). LR-CD syntheses at alkaline pH or at a long incubation time favoured low-degree of polymerization (DP) products (CD22-CD32). Addition of 5-15% dimethyl sulfoxide (DMSO) promoted the synthesis of medium-DP species (CD33-CD43) by 10-25%. Pretreatment of pea starch with isoamylase could enhance the yield of LR-CDs. The ratio of LR-CD products depends on pH, incubation time and addition of organic solvents such as DMSO. LR-CD yield can be increased by thorough optimization of starch types, starch concentrations, enzyme activities, pH and incubation times. This study is the first report of the effect of organic solvents on LR-CD production by amylomaltase.

Synthesis of large-ring cyclodextrin from tapioca starch by amylomaltase and complex formation with vitamin E acetate for solubility enhancement

Recombinant amylomaltase from Corynebacterium glutamicum (CgAM) was used to synthesize large-ring cyclodextrins (LR-CDs) from tapioca starch. A mixture of LR-CDs (CD22–CD54) was obtained from both raw and soluble tapioca starch, but with different ratio of main products (CD25–CD30). Almost three-fold higher amount of products was formed indicating the preference for raw tapioca starch. The optimal condition was to incubate 1.0% (w/v) starch with 0.05U/ml CgAM in buffer pH 6.0 at 30°C for 18h. Total LR-CDs yield was 60.5mg/g starch. LR-CDs were resistant to hydrolysis by Rhizopus sp. glucoamylase but completely hydrolyzed by CgAM at high enzyme concentration. Solid inclusion complex between LR-CDs mixture and vitamin E acetate was formed, as analyzed by FTIR. For complex preparation, the 1:10mol ratio of LR-CDs to vitamin E was chosen because maximum amount of the vitamin was bound to LR-CDs and high complex yield was obtained. Aqueous solubility of vitamin E acetate in complex form increased 800-fold while the antioxidant activity decreased about 30%. This is the first work to demonstrate the use of tapioca starch in LR-CDs synthesis by CgAM and the complex formation between LR-CDs and vitamin E acetate as solubility enhancement process of vitamin.

Multi-wavelength colorimetric determination of large-ring cyclodextrin content for the cyclization activity of 4-α-glucanotransferase

Large-ring cyclodextrins (LR-CDs) have a number of intriguing properties for potential use in pharmaceutical and food industry. To date, no colorimetric method has been reported for LR-CD content quantification. In this study, triple wavelength colorimetry (TWC) and orthogonal-function spectrophotometry (OFS) have been successfully applied to determine ingredient concentrations in a mixture of amylose and LR-CDs. Both TWC and OFS yielded precise amylose content data in good agreement with expected values. For quantification of LR-CD content, OFS provided a higher accuracy than TWC, which resulted in a slight over-determination. As a comparison, single-wavelength colorimetry performed at the corresponding absorption maximum led to a significant over-determination of both amylose and LR-CD contents. The validity of TWC and OFS allowed their application for discriminative detection of the cyclization and total activity of a 4-α-glucanotransferase (4αGTase) from Thermus aquaticus regarding the synthesis of LR-CDs and the conversion of amylose to small molecules, respectively. High pressure size exclusion chromatography analysis of the post-reaction mixtures following 4αGTase-catalyzed conversion of amylose revealed the presence of linear malto-oligosaccharides in the LR-CD fraction. By introduction of a correction factor, the interference caused by linear malto-oligosaccharides was eliminated for a more accurate determination of LR-CD cyclization activity.

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.

Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production

The aim of this study was to isolate a novel amylomaltase gene from community DNA of soil samples collected from Ban Nong Khrok hot spring in Thailand without bacterial cultivation. Using PCR, a 1.5 kb full-length gene was amplified and ligated with pGEM-T easy vector to transform into Escherichia coli DH5 alpha for sequencing. The obtained gene encoding an amylomaltase consisted of 1.503 bp that translated into 500 amino acids. Amino acid sequence deduced from this gene was highly homologous with that of amylomaltase from Thermus thermophillus ATCC 33923. In order to express the enzyme, the cloned gene was subcloned into plasmid pET-17b and introduced into E. coli BL21 (DE3). The maximum expression was observed when the cloned cells were cultured at 37 degrees C for 6 h with 0.5 mM IPTG induction. By 10% SDS-PAGE, the relative molecular mass of the purified amylomaltase was approximately 58 kDa. This enzyme was optimally active at 70 degrees C and pH 9.0. In addition, the enzyme could hydrolyze pea starch to yield the large-ring cyclodextrins with degrees of polymerization of 23 and higher. It is noted that CD29 was the product in the largest quantity under all tested conditions.

Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production

Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production