Effect of organic solvents on the yield and specificity of cyclodextrins by recombinant cyclodextrin glucanotransferase (CGTase) from Anaerobranca gottschalkii

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an enzyme that degrades starch and starch related glucans into cyclodextrins (CDs) by intramolecular transglycosylation reaction. The biochemical activity of recombinant CGTase from Anaerobranca gottschalkii for the yield and product specificity of cyclodextrins was investigated in the presence of organic solvents. Compared with the control of starch bioconversion, addition of various organic solvents generally increased the total CD and product specificity by affecting product inhibition and/or intermolecular transglycosylation reaction. The highest conversion (45 %) of starch to CDs was obtained in the presence of ethanol, while the simultaneous addition of two organic solvents, decanol-ethanol, comparatively showed a reduced total yield of 39 %. Despite this, the highest product ratio of 91 % α-CD, and 64 % β-CD was obtained in the presence of decanol and cyclohexane respectively. The effect of organic solvents on the yield and specificity of CD was attributed mainly to their effect on product inhibition and transglycosylation reaction. Although the use of two organic solvents showed almost a significant increase in total yield of CDs, it resulted in a comparatively lower specific product yield compared to their respective individual effect. Generally, normal enzyme activity was favoured at higher temperature of 65 °C, but the addition of organic solvents, in most cases, was found to decrease the bioconversion. Thus, the preferred optimal condition was reduced to 40 °C, where the maximal conversion of starch to CDs in general and α-CD in particular was achieved.     

Enhanced conversion of starch to cyclodextrins in ethanolic solutions by bacillus circulans var alkalophilus cyclomaltodextrin glucanotransferase

Improved formation of cyclodextrins (CDs) from starch in ethanolic solutions byBacillus circulans var alkalophilus cyclomaltodextrin glucanotransferase was studied. The β- and γ-CD yields increased and α-CD yield gradually decreased as the ethanol concentration was raised. The ethanol concentration required for maximal CD yield depended essentially on starch concentration. The ethanol's effect was pronounced at high starch concentrations. For example, with 30% (w/v) starch, the CD yield was 2.4-fold (146.5 g/L) in the presence of 15% (v/v) ethanol. The effect of dimethylsulfoxide on the formation of CDs was similar to that of ethanol. The disintegration of β- and γ-CDs were narrowly interdependent on the formation of a α-CD and malto-sugars. The amount of reducing sugars decreased from a dextrose equivalent value of roughly 7.5 to 4.5 in the presence of ethanol at starch concentrations 1-30% (w/v). The effect of ethanol on starchy materials from various sources was similar. It was concluded that ethanol retards the decomposition of β-CD by a general mechanism involving a decreased activity of water.     

Evaluation the effect of cyclodextrin complexation on aqueous solubility of fluorometholone to achieve ophthalmic solution

In this study, the effect of different CDs including α-CD, β-CD, γ-CD, hydroxypropyl β-CD (HP β-CD), sulphobutylether β-CD (SBE β-CD) and HP γ-CD on aqueous solubility of fluorometholone (Flu) was investigated. Also the phase solubility studies were performed in the presence of eye drop excipients such as benzalkonium chloride, hydroxypropyl methylcellulose (HPMC) and buffers. The aqueous solubility of Flu was increased by 8, 15, 5, 100, 65 and 135 folds in the presence of 20% w/v α-CD, β-CD, γ-CD, HP β-CD, HP γ-CD and SBE β-CD, respectively. Aqueous solubility of Flu was 0.43 ± 0.08 and 1.16 ± 0.04 mg/mL in systems containing 5% w/v HP γ-CD and SBE β-CD, respectively. The aqueous solubility of Flu in the presence of HP γ-CD was not influenced by buffer type while the phosphate buffer caused a reduction in the aqueous solubility in the presence of SBE-β-CD. Also, investigations on the solubility of Flu in water in the presence of 5% HP γ-CD and SBE-β-CD and the additives such as benzalkonium chloride and HPMC indicated that these components had no remarkable effect on the aqueous solubility of Flu. In conclusion, CD complexation is able to improve the aqueous solubility of Flu and it would be possible to prepare ophthalmic solution of Flu by this method.     

Effects of water and alcohol on the formation of inclusion complexes of d-limonene and cyclodextrins

Abstract

Systematic studies have been carried out on the role of water and alcohol in the formation of inclusion complexes between d-limonene and α-, β- and γ-cyclodextrin (CD) by a micro-aqueous method. The inclusion complex was barely formed at zero water content for all CDs. Above the specific water content for each CD, formation of the inclusion complex correlated well with an equation which was derived on the autocatalytic assumption for the inclusion phenomenon. The inclusion complex correlated well with an equation which was derived on the autocatalytic assumption for the inclusion phrnomenon. The minimum water content, which was defined as 1% of the maximum concentration of the inclusion complex formed, coincided with the number of water molecules inside the cavity of the CD. In the presence of ethanol, a significant amount of the inclusion complex was formed for β- and γ-CD/limonene systems, particularly at lower moisture content. However, for α-CD the inclusion fraction decreased significantly in the presence of ethanol. This means that ethanol inhibits the formation of the inclusion complex between x-CD and d-limonene. For other linear alcohols, the formation of the inclusion complex between d-limonene and β-CD increased with decreasing alkyl chain length. This suggests that the more hydrophilic and the smaller (in molecular size) the alcohol is, the more enhanced is the inclusion of d-limonene to β-CD.     

Studies of cyclodextrin effect for glycosylation by galactosyltransferase

Cyclodextrins (CDs) do not disturb HPLC purification and do not form lather, which makes them superior to commonly used surfactants. In this paper, the authors describe the utility of CDs, specifically α-CD, β-CD, and γ-CD, for the synthesis of neoglycolipids from insoluble peptide-linked substrates. γ-CD was found to be well matched to the bulky benzyl group and accelerated the reaction for the substrate having Phe (F), while β-CD was well matched to the methyl group and accelerated the reaction for the substrate having Ala (A). Galactosylation to water-insoluble peptide-linked glucosaminide substrate by galactosyltransferase proceeded 63% at the most, but using either β-CD or γ-CD gave quantitative yield within 10 h.     

The effect of cyclodextrin mixtures on aqueous solubility of beclomethasone dipropionate

The aim of present study was to evaluate the effect of natural, synthetic cyclodextrins (CDs) and CD mixtures on aqueous solubility of beclomethasone dipropionate (BDP). The phase solubility studies were done in the presence of 6 CDs. Furthermore, aqueous solubility of BDP was tested in the presence of CD mixtures. The solubility of BDP in water was increased by 30, 77, 155 and 30 folds in the solution containing 20%?w/v α-CD, hydroxylpropyl β-CD (HP-β-CD), hydroxypropyl γ-CD (HP-γ-CD) and sulphobutylether β-CD (SBE-β-CD), respectively. CD mixtures had remarkable effect on the aqueous solubility of BDP so that solubility in water increased between 200 and 1,500 times in the presence of different CD mixtures. Further addition of sodium acetate to the solubilisation medium reduced the aqueous solubility. In conclusion, CD complexation was able to improve the aqueous solubility of BDP. The synergistic effect of cyclodextrin mixture was observed.     

Electrospinning of cyclodextrin functionalized polyethylene oxide (PEO) nanofibers

By means of the electrospinning technique we have successfully synthesized cyclodextrin (CD) functionalized polyethylene oxide (PEO) nanofibers (PEO/CD) with the ultimate goal to develop functional nanowebs. Three different types of CDs; α-CD, β-CD and γ-CD are incorporated individually in electrospun PEO nanofibers. The aqueous solutions containing different amount of PEO (3%, 3.5% and 4% (w/v), with respect to solvent) and CDs (25% and 50% (w/w), with respect to PEO) are electrospun and bead-free nanofibers are obtained. The presence of the CDs in the PEO solutions is found to facilitate the electrospinning of bead-free nanofibers from the lower polymer concentrations and this behavior is attributed to the high conductivity and viscosity of the PEO/CD solutions. The presence of CDs in the electrospun PEO nanofibers is confirmed by Fourier transform infrared (FTIR) spectroscopy studies. The 2-D X-ray diffraction (XRD) spectra of PEO/CD nanowebs did not show any significant diffraction peaks for CDs indicating that the CD molecules are distributed within the polymer matrix without any phase separated crystalline aggregates.     

Design on head-to-tail directly linked homogeneous and heterogeneous cyclodextrin dimers and their evaluation of hydrophobic cavity

Covalent directly head-to-tail linked homogeneous and heterogeneous cyclodextrin (CD) dimers were synthesized, and that the reaction of 6-tosylated α-, β-, or γ-CD with a β-CD mono-oxyanion linked the second CD to the secondary hydroxyl side of β-CD was demonstrated. Moreover, deprotonation of α- and γ-CD using NaOH gave corresponding mono-oxyanions, which reacted with the 6-tosylated CDs to produce the CD dimers. The binding of the dimers to sodium 6-(4-tert-butylaniline)-2-naphthalenesulfonate (BNS) was investigated. The binding constant of the ⁶β–²β-CD dimer with BNS was estimated as 3.2 × 10⁶ M⁻¹, about 10² times larger than that of β-CD monomer.     

The cavity size effect on the fluorescence properties of 4′-dimethylaminoacetophenone complexed with cyclodextrins

Fluorescence properties of excited 4^′-dimethylaminoacetophenone (DMAAP) complexed with α-, β-, and γ-cyclodextrins (CDs) were studied by means of steady state and time-resolved laser spectroscopy. The 1:2 DMAAP–α-CD and 1:1 DMAAP–β-CD complexes exhibited dual fluorescence in neutral aqueous solutions while only the fluorescence from the locally excited state was observed in the case of DMAAP complexed with γ-CD. The CD cavity size effect on the excited state dynamics of DMAAP–CD complexes was further discussed. It revealed that polarity effect introduced by the hydrophobic cavity is more important in controlling of the photochemistry of DMAAP than the restriction of molecular motion inside the CD cavity.     

Photophysics of a cyanine dye within cyclodextrin cavity

The modulated photophysical and dynamical behavior of a potent anti-tumor photosensitizer 3,3^/-diethyloxadicarbocyanine iodide (DODCI) following host-guest inclusion complex formation with α-, β- and γ-Cyclodextrins (CDs) has been studied using steady-state and time-resolved spectroscopic methods. The cavity size of the CDs (α-CD <β-CD <γ-CD) is argued to play an instrumental role underlying the formation of the host-guest inclusion complex. While negligible interaction with α-CD is found to be succeeded by prominent quenching of monomeric fluorescence of the dye within β-CD and γ-CD with the degree of quenching being greater within γ-CD. The most appealing fact attained from the experimental results is the anticipation of dimer formation of DODCI within the large cavity of γ-CD which can entrap more than one molecule of DODCI. The steady-state results are found to be adequately corroborated by time-resolved fluorescence decay studies. Such encapsulation of the cyanine dye within the carrier cargo can be designed for targeted delivery inside biological systems.     

Spectral modulation of a charge transfer reaction of 2-methoxy-4-(N,N-dimethylamino)benzaldehyde inside cyclodextrin nanocage

This article reports modulation of intramolecular charge transfer (ICT) reaction of 2-methoxy-4-(N,N-dimethylamino)benzaldehyde (2-MDMABA) encapsulated within the cyclodextrin nanocavities investigated by steady state and time resolved measurements. The ICT emission, absent in bulk water, originates in the presence of α-, β- and γ-CD with the huge enhancement of local emission. From the Benesi–Hildebrand plot, the stoichiometry of the host–guest inclusion complex is found to be 1:1 for β- and γ-CD whereas 1:1 and 1:2 guest to host complexation occur at low and high concentration of α-CD, respectively. The association constants of the inclusion complexes have also been estimated from the Benesi–Hildebrand plot. The greater binding capability of 2-MDMABA with β-CD than that of other two CDs is further supplemented by time resolved study.     

Gamma-cyclodextrin on enhancement of water solubility and store stability of nystatin

The water solubility of nystatin was found enhanced by forming inclusion complex with gamma-cyclodextrin (γ-CD). Further discovery of a pleased surprise showed that the phase solubility curves of nystatin in β- and γ-CD aqueous solution were A_L type, while B_S type for α-CD, indicating 1:1 inclusion complexes were formed between β-CD, γ-CD and nystatin, but no inclusion complexes for α-CD, in addition, CDs with much larger ring would be more suitable for forming inclusion complexes with macrolide antibiotics. The aqueous solubility of nystatin in γ-CD solution was investigated increased with γ-CD concentration increasing. At the concentration of 24 g/100 ml for γ-CD aqueous solution, which is near to the saturated solution, water solubility of nystatin was found to be 104 μg/ml, which was 103 folds over original nystatin. Inclusion constants for γ-CD–nystatin complexes were 0.539 l/mmol, which is larger than that of β-CD–nystatin complex (0.375 l/mmol). The inclusion complex of γ-CD with nystatin was prepared and detected by infrared spectrum, results showing that the ester linkage and diene were included in the cavity of CDs, while conjugate arachidonic, carboxyl and amino group were left outside of CDs. Storing experiment showed that forming of the inclusion complexes greatly enhanced the stability of nystatin against light and oxygen.     

Separation of terbutaline enantiomers in capillary electrophoresis with neutral cyclodextrin-type chiral selectors and investigation of the structure of selector-selectand complexes using nuclear magnetic resonance spectroscopy

The major goal of this study was to determine the affinity pattern of the terbutaline (TB) enantiomers toward α-, β-, γ-, and heptakis(2,3-di-O-acetyl)-β-cyclodextrins and using NMR spectroscopy for the understanding of the fine mechanisms of interaction between the cyclodextrins (CD) and TB enantiomers. It was shown once again that CE in combination with NMR spectroscopy represents a sensitive tool to study the affinity patterns and structure of CD complexes with chiral guests. Opposite affinity patterns of TB enantiomers toward native α- and β-CDs were associated with significant differences between the structure of the related complexes in solution. In particular, the complex between TB enantiomers and α-CD was of the external type, whereas an inclusion complex was formed between TB enantiomers and β-CD. One of the possible structures of the complex between TB and heptakis(2,3-di-O-acetyl)-β-CD (HDA-β-CD) was quite similar to that of TB and β-CD, although the chiral recognition pattern and enantioselectivity of TB complexation with these two CDs were very different.     

Formation of nanotubes and secondary assemblies of cyclodextrins induced by BBOT

The study of cyclodextrin nanotubes is a significant topic among the self-assembly behaviors of cyclodextrins. We report herein the interaction of 2,5-bis(5′-tert-butyl-2-benzoxazoyl)thiophene (BBOT) with α-, β-, γ-cyclodextrins (CDs). It has been discovered that the reaction patterns of BBOT with CDs are remarkably different. β-CD forms a simple inclusion complex with BBOT in a stoichiometry of 1:2 (guest:host). β-CD forms a 1:1 inclusion complex with BBOT at its low concentration. At higher concentration of BBOT, the nanotube and secondary assembly of β-CD are formed. As for γ-CD, the nanotube and secondary assembly are formed within the whole concentration range of BBOT studied. The structure of γ-CD nanotubes is different from that of β-CD nanotubes to a certain extent.

     

Effect of the ring size and asymmetry of cyclodextrins on their inclusion ability: a theoretical study

Effect of the ring size and asymmetry upon methylation of cyclodextrins (CDs) on their inclusion ability has been demonstrated for the inclusion complexes of native α-, β-, γ-CDs, dimethylated β-CD (DIMEB) and trimethylated β-CD (TRIMEB) with piperazine (PIZ) by PM3 and ONIOM calculations. In all complexes, PIZ prefers residing mostly in the central CD cavity. The complex stability in the order TRIMEB–PIZ > DIMEB–PIZ > α-CD–PIZ > γ-CD–PIZ > β-CD–PIZ indicates that the CD-ring asymmetry promotes the macrocycle deformation and inclusion ability. Our calculation results suggest that the inclusion complexes of both native and methylated CDs with PIZ in the gas phase are energetically stable, in addition to the β-CD–PIZ inclusion complex that has been evidenced thus far by X-ray crystallographic and spectroscopic analyses. Further calculations in the presence of water and adjacent CD molecules show that the increased intermolecular hydrogen bond interactions enhance the stability of β-CD–PIZ complex.     

Basic hydrolysis of carbofuran in the presence of cyclodextrins

The influence of cyclodextrins (CDs; α-CD, β-CD and γ-CD) upon the basic hydrolysis of carbofuran (CF) was studied. The observed behaviour was an inhibition and this decrease in the rate constants is due to the formation of an unreactive complex between CF and the CDs. A kinetic model was applied to this system and the kinetic coefficients were obtained.     

Separation of enantiomers of ephedrine by capillary electrophoresis using cyclodextrins as chiral selectors: comparative CE, NMR and high resolution MS studies

The enantiomer migration order (EMO) of ephedrine was investigated in the presence of various CDs in CE. The molecular mechanisms of chiral recognition were followed for the ephedrine complexes with native α- and β-CD and heptakis(2,3-di-O-acetyl-6-O-sulfo)-β-CD (HDAS-β-CD) by CE, NMR spectroscopy and high-resolution MS. Minor structural differences were observed between the complexes of ephedrine with α- and β-CD although the migration order of enantiomers was opposite when these two CDs were applied as chiral selectors in CE. The EMO was also opposite between β-CD and HDAS-β-CD. Significant structural differences were observed between ephedrine complexes with the native CDs and HDAS-β-CD. The latter CD was advantageous as chiral CE selector not only due to its opposite electrophoretic mobility compared with that of the cationic chiral analyte, but also primarily due to its enhanced chiral recognition ability towards the enantiomers of ephedrine.     

Purification and Properties of a New Thermostable Cyclodextrin Glucanotransferase from Bacillus pseudalcaliphilus 8SB

A new cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkaliphilic halotolerant Bacillus pseudalcaliphilus 8SB was studied in respect to its γ-cyclizing activity. An efficient conversion of a raw corn starch into only two types of cyclodextrins (β- and γ-CD) was achieved by the purified enzyme. Crude enzyme obtained by ultrafiltration was purified up to fivefold by starch adsorption with a recovery of 62% activity. The enzyme was a monomer with a molecular mass 71 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The CGTase exhibited two pH optima, at pH 6.0 and 8.0, and was at most active at 60 °C and pH 8.0. The enzyme retained more than 80% of its initial activity in a wide pH range, from 5.0 to 11.0. The CGTase was strongly inhibited by 15 mM Cu(2+), Fe(2+), Ag(+), and Zn(2+), while some metal ions, such as Ca(2+), Na(+), K(+), and Mo(7+), exerted a stimulating effect in concentration of 5 mM. The important feature of the studied CGTase was its high thermal stability: the enzyme retained almost 100% of its initial activity after 2 h of heating at 40-60 °C; its half-life was 2 h at 70 °C in the presence of 5 mM Ca(2+). The achieved 50.7% conversion of raw corn starch into 81.6% β- and 18.4% γ-CDs after 24 h enzyme reaction at 60 °C and pH 8.0 makes B. pseudalcaliphilus 8SB CGTase industrially important enzyme for cyclodextrin production.     

Chemical and enzymatic synthesis of neoglycolipids in the presence of cyclodextrins

The efficiency of cyclodextrins (CDs), α-CD, β-CD and γ-CD, for the blotting of hydrophobic substrates to water-soluble polymers and for the synthesis of neoglycolipids using glycosyltransferase is described. CDs did not disturb HPLC purification and did not bring lather. These merits are superior to surfactants commonly used in such a case. The utility of CDs as a novel and efficient supporting material for enzymatic glycosylation is also discussed.     

Cyclodextrin induced switch between heterolytic and homolytic dediazoniation mechanisms

We have investigated the kinetics and mechanism of dediazoniation of 4-nitrobenzenediazonium (4NBD) tetrafluoroborate in the presence of α-cyclodextrin, α-CD, and γ-cyclodextrin, γ-CD, under acidic (HCl, pH=2) conditions by employing a combination of spectrometric and chromatographic techniques. In the absence of CDs, dediazoniation follows first-order kinetics, with t(1/2)=22,000 s at T=60 °C, but addition of small amounts of either α-CD or γ-CD leads to more rapid but not first-order kinetics with t(1/2)~400 s when [α-CD]=20 [4NBD] or [γ-CD]=15 [4NBD]. Analyses of reaction mixtures by HPLC indicate that three main dediazoniation products are formed depending on the particular experimental conditions. These are 4-nitrophenol, ArOH, nitrobenzene, ArH, and 4-nitrochlorobenzene, ArCl. In the absence of CDs, the main dediazoniation product is the substitution product ArOH, but on increasing the concentration of CD, the reduction product ArH becomes predominant at the expense of ArOH, indicating that a switch between the heterolytic and homolytic reaction mechanisms take place under acidic conditions, where little significant ionization of the OH groups of the CDs takes place (pK(a)≈12). Addition of the surfactant sodium dodecylsulfate, SDS, blocks the CD cavity inhibiting 4NBD dediazoniation and decreasing the yields of ArH with a concomitant increase in that of ArOH, suggesting that 4NBD ions form an inclusion complex prior to reacting with the OH groups of the CDs. This O-coupling reaction leads to the formation of a highly unstable Z-diazo ether adduct that cannot isomerize to the much more stable E-isomer because of the geometric restrictions imposed by the CD cavity, splitting homolitically.