Production and purification of CGTase of alkalophylicBacillus isolated from Brazilian soil

Alkalophylic bacilli that produce cyclodextringlycosyltransferase (CGTase) were isolated from Brazilian soil, with a scheme of two plating steps. In the first step, the bacterial isolate forms a halo in the cultivation medium that contains γ-cyclodextrin (CD) complexing dyes. The CGTase of an isolate was purified 157-fold by biospecific affinity chromatography, with β-CD showing a mol wt of 77,580 Daltons. It produces a γ- to β-CD ratio of 0.156 and a small amount of α-CD, using maltodextrin 10% as substrate, at 50°C, pH 8.0 and 22 h reaction time, reaching 21.4% conversion of the substrate to cyclodextrins. In the second screening step, the isolates chosen give larger halos with β-CD complexing dyes, and smaller halos with β-CD complexing dyes, leading to a 30% improvement in γ-CD selectivity, although at lower total yield for cyclodextrins (11.5%).     

Inclusion complexation of diclofenac with natural and modified cyclodextrins explored through phase solubility, 1H-NMR and molecular modeling studies

Guest–host interactions were examined for neutral diclofenac (Diclo) and Diclofenac sodium (Diclo sodium) with each of the cyclodextrin (CD) derivatives: α-CD, β-CD, γ-CD and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), all in 0.05 M aqueous phosphate buffer solution adjusted to 0.2 M ionic strength with NaCl at 20 °C, and with β-CD at different pHs and temperatures. The pH solubility profiles were measured to obtain the acid–base ionization constants (pK _as) for Diclo in the presence and absence of β-CD. Phase solubility diagrams (PSDs) were also measured and analyzed through rigorous procedures to obtain estimates of the complex formation constants for Diclo/CD and Diclo sodium/CD complexation in aqueous solutions. The results indicate that both Diclo and Diclo sodium form soluble 1:1 complexes with α-, β-, and HP-β-CD. In contrast, Diclo forms soluble 1:1 Diclo/γ-CD complexes, while Diclo sodium forms 1:1 and 2:1 Diclo/γ-CD, but the 1:1 complex saturates at 5.8 mM γ-CD with a solubility product constant (pK _sp = 5.5). Therefore, though overall complex stabilities were found to follow the decreasing order: γ-CD > HP-β-CD > β-CD > α-CD, some complex precipitation problems may be faced with aqueous formulations of Diclo sodium with γ-CD, where the overall concentration of the latter exceeds 5.8 mM γ-CD. Both ¹H-NMR spectroscopic and molecular mechanical modeling (MM⁺) studies of Diclo/β-CD indicate the possible formation of soluble isomeric 1:1 complexes in water.     

Characterization of cyclodextrin glycosyltransferase from Bacillus firmus strain No. 37

The enzyme cyclod extringly cosyltransferase (CGTase), EC2.4.1.19, which produces cyclodextrins (CDs) from starch, was obtained from Bacillus firmus strain no. 37 isolated from Brazilian soil and characterized in the soluble form using as substrate 100 g/L of maltodextrin in 0.05 M Tris-HCl buffer, 5 mM CaCl₂, and appropriate buffers. Enzymatic activity and its activation energy were determined as a function of temperature and pH. The activation energy for the production of β- and γ-CD was 7.5 and 9.9 kcal/mol, respectively. The energy of deactivation was 39 kcal/mol. The enzyme showed little thermal deactivation in the temperature range of 35–60°C, and Arrhenius-type equations were obtained for calculating the activity, deactivation, and half-life as a function of temperature. The molecular weight of the enzyme was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, giving 77.6k Da. Results for CGTase activity as a function of temperature gave maximal activity for the production of β-CD at 65°C, pH 6.0, and 7 1.5 mmol of β-CD/(min·mg of protein), whereas for γ-CD it was 9.1 m mol of γ-CD/(min·mg of protein) at 70°C and pH 8.0. For long contact times, the bestuse of the enzymatic activity occurs at 60°C oratalower temperature, and the reaction pH may be selected to increase the vield of a desired CD.     

Enhancement of selectivity for producing γ-cyclodextrin

The production of cyclodextrins (CDs) by cyclodextrin-glycosyl-transferase (CGTase) from Bacillus firmus was studied, with respect to the effect of the source of starch upon CD yield and on the selectivity for producing γ-CD. Cyclodextrin production tests were run for 24 h at 50°C, pH 8.0, and 1 mg/L of CGTase, and substrates were maltodextrin or the starches of rice, potato, cassava, and corn hydrolyzed up to D. E. 10. Cornstarch was the best substrate for producing γ-CD. Later, glycyrrhizin (2.5% [w/v]), which forms a stable complex with γ-CD, was added to the cornstarch reaction medium and increased the yield of γ-CD to about four times that produced with only maltodextrin, but the total yield of CDs remained practically unchanged. Therefore, the results showed that the studied CGTase is capable of giving relatively high yield of γ-CD in the presence of glycyrrhizin as complexant and cornstarch as substrate.     

Elucidation of the Role of the Complex in Hydride Transfer Reaction between Methylene Blue and 1-Benzyl-1,4-dihydronictinamide by Effect of γ-Cyclodextrin

The kinetics of the hydride transfer reaction between Methylene Blue (MB⁺) and 1-benzyl-1,4-dihydronicotinamide (BNAH) were studied in 10% ethanol-90% water mixed solvents containing β- and γ-cyclodextrins (β-CD and γ-CD). The pseudo-first order rate constant shows kinetic saturation at high initial concentration of BNAH. This indicates the formation of a complex between MB⁺ and BNAH. The reaction was suppressed by addition of β-CD, but enhanced by addition of γ-CD. MB⁺ and BNAH were separately accommodated within the β-CD cavity and the cavity walls may protect the activity site of the reactants. On the other hand, in the MB⁺-BNAH-γ-CD system, the inclusion of the complex between MB⁺ and BNAH with γ-CD occurred. This effect of γ-CD can distinguish between the productive and non-productive nature of the complex.     

Investigation of Drug Nanoparticle Formation by Co-grinding with Cyclodextrins: Studies for Indomethacin, Furosemide and Naproxen

Drugs with poor water solubility were co-ground with cyclodextrins (CDs) to create nanoparticles with improved solubility characteristics. Indomethacin (IDM), furosemide (FRM) and naproxen (NAP) were co-ground with β-CD at the molar ratio of 2:1 (CD:drug). Co-grinding of a drug with CD resulted in not only the formation of drug nanoparticles but also the solubilization of the drug by inclusion complex formation with CD in aqueous media. The nanoparticle fraction of IDM, and FRM from ground mixtures prepared with β-CD was as high as 60–70% while the solubilization fraction was less than 10%. In contrast, β-CD–NAP ground mixture showed a large fraction, 48%, for drug solubilization and only 4% for nanoparticle formation. Furosemide ground mixtures prepared with α-CD, β-CD and γ-CD showed comparatively high nanoparticle fraction while the solubilization fraction was around 10%. Both the nanoparticle fraction and the solubilization fraction were greater in the IDM–β-CD system than those in γ-CD and α-CD systems. The nanoparticle formation of NAP depended on the types of CD used as a co-grinding additive. Naproxen nanoparticles could be prepared by co-grinding NAP and α-CD, while the solubilization of NAP tended to improve when β-CD or γ-CD was used.     

Thermodynamic evaluation of antibacterial activity for inclusion complexes of amoxicillin with cyclodextrins

The antibacterial action of amoxicillin (AMPC) and the inclusion complexes of AMPC with α-, β- and γ-cyclodextrins (α-CD, β-CD and γ-CD, respectively) to Escherichia coli B (E. coli) was evaluated by isothermal titration microcalorimetry and by petri-dish bioassay method. The effects of the compounds on produced heat during the exponential phase of the E. coli growing were measured and the growing rate constants of the cells was calculated from the power-time (p-t) curve before and after the treatment with AMPC. Results from the both methods showed that the antibacterial activity became stronger in the following order: AMPC-βCD > AMPC-γCD ≈ AMPC-αCD > AMPC only.     

Inclusion complexes of fusidic acid and three structurally related compounds with cyclodextrins

The inclusion complexes between fusidate, 3-keto fusidate, 11-keto fusidate and 11-deoxy fusidate and α-, β-, and γ-cyclodextrin (CD) were studied using capillary electrophoresis. By monitoring the changes in mobility of the negatively charged compounds in the presence of varying amount of CD the stability constants of the complexes formed could be obtained. In the case of α- and β-CD the obtained results could be modelled to a simple model assuming 1:1 stoichiometry, revealing, not surprisingly, that β-CD formed a stronger complex compared to α-CD. A model assuming 1:2 (fusidate:CD) stoichiometry could be fitted to the data obtained with γ-CD. The results showed that the different fusidanes formed very strong 1:1 complexes with γ-CD as well as a quite weak 1:2 complex. 3-keto-, 11-keto- and 11-deoxy-fusidate formed stronger complexes compared to fusidate, probably due to an decrease in hydrophilicity caused by the reduced number of hydroxyl groups. The complex between γ-CD and fusidate was studied by use of 2D-NMR spectroscopy. The results showed that most of the hydrogen atoms of fusidate show interactions with the hydrogen atoms in the cavity of γ-CD. The interaction pattern suggests that fusidate may be fully embedded in the cavity of γ-CD. No interactions between fusidate and the hydrogen atoms situated at the outside of the CD were found.     

Comparative Study of the Inclusion Complexation of Pizotifen and Ketotifen with Native and Modified Cyclodextrins

The interaction of two benzocycloheptanes namely, pizotifen (Pizo) and ketotifen (Keto), with cyclodextrins (CDs: α-, β-, γ-, and HP-β-CDs) has been investigated by several techniques including phase solubility, X-ray powder diffractometry, ¹H-nuclear magnetic resonance and molecular mechanical modeling. The effects of CD type, pH, ionic strength and temperature on complex stability were also explored. The complex formation constant (K ₁₁) values for the Pizo/CD system follows the decreasing order β-CD > γ-CD > HP-β-CD > α-CD. However, for the Keto/CD system it follows the decreasing order γ-CD > β-CD > HP-β-CD > α-CD. The tendency of Pizo and Keto to complex with β-CD is driven to the extent of 70% by the hydrophobic effect. Complex formation of Keto and Pizo was substantially driven by entropy (>100 J⋅mol⁻¹⋅K⁻¹) but slightly retarded by enthalpy (3–8 kJ⋅mol⁻¹). ¹H-NMR and MM⁺ studies indicate multimodal inclusion of the methylpiperadine, thiophene and phenyl moieties into the β-CD cavity.     

Thermal decomposition behaviors of β-cyclodextrin,its inclusion complexes of alkyl amines,and complexed β-cyclodextrin at different heating rates

The present work revealed there was a conceptual difference in the thermal decomposition behaviors between the complexed β-cyclodextrin (CD) in an inclusion system and the β-CD complex of guest. The thermal decomposition behaviors of the solid inclusion complexes of β-CD with ethylenediamine (Eda), diethylenetriamine (Dta) and triethylamine (Tea) were investigated using nonisothermal thermogravimetry (TG) analysis based on weight loss as a function of temperature. In view of TG profiles, a consecutive mechanism describing the formation and thermal decomposition of the three solid supermolecules of β-CD was presented. Heating rate has very different effects on the thermal decomposition behaviors of these complexes. The faster the heating rate is, the higher the melting-decomposition point of the complexed β-CD in an inclusion system is, and on the whole the bigger the rate constant (k) of the thermal decomposition reaction of the complexed β-CD is. The thermal decomposition process of the complexed β-CD for each inclusion system is determined to be simple first-order reaction using Ozawa method. The apparent activation energies (E _a) and frequency factors (A) of the thermal decomposition reactions of the complexed β-CD molecules have been also calculated. It is found that when the decomposition reaction of the complexed β-CD encountered a large value of E _a, such as that in Dta–β-CD system, an apparent compensation effect of A on E _a can provide enough energy to conquer the reaction barrier in prompting the k value of thermal decomposition reaction of the complexed β-CD according to Arrhenius equation.     

Cyclodextrin effect on solvolysis of ortho benzoyl chlorides

A kinetic study was carried out on the solvolysis of ortho benzoyl chlorides in the presence of α-, β- and γ-Cyclodextrin (CD). The solvolysis mechanism of benzoyl chlorides is sensitive to the substituents, and to the solvent in which the reaction takes place. In water, the behaviour exhibited by benzoyl chlorides which have electron-attracting groups, is consistent with an associative mechanism whilst electron-donating substituents induce a dissociative mechanism. The results obtained in the presence of CD show a decrease in the observed rate constant, k _obs, as the CD concentration increases. This behaviour can be explained if these substrates undergo solvolysis through a dissociative path in the presence of α-, β- and γ-CD.     

Inclusion complexes of spin-labeled indoles with cyclodextrins in aqueous solutions

Guest-host complexes of β- and γ-cyclodextrins (CDs) with two spin-labeled indole derivatives having the same molecular weights but different structures were studied by EPR spectroscopy in aqueous solutions and semiempirical quantum-chemical calculations of these systems were carried out. In the presence of CD the polarity of the NO group environment decreases and the rotational correlation time (τ) of guest molecules increases. Both indole derivatives form 1 : 1 complexes with γ-CD, the binding constants of the complexes being different more than twice. Simulation of EPR spectra made it possible to determine the indole ring orientation relative to the plane of the host molecule (at angles in the range 30–60°) and the rotational diffusion coefficients of the complexes, which corresponded to the hydrodynamic volume of one γ-CD molecule. In contrast to the complexes with γ-CD the rotational correlation times, τ, of the complexes with β-CD correspond to a hydrodynamic volume which much exceeds the volume of a single β-CD molecule. The complexes with β-CD are also characterized by more hydrophobic environment for guest molecules and absence of spin exchange with Ni²⁺ ions in the aqueous solution. There results are consistent with a dimeric structure of β-CD in the complex and with the orientation of the long axis of the guest molecule along the dimer axis. The energies and geometric parameters were calculated for all complexes by the PM3 method with a conventional set of parameters. The optimized energetically stable structures of the 1 : 1 complexes with γ-CD and of the 1 : 2 complexes with β-CD are consistent with experimental data. __________ Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1139–1147, May, 2005.     

Production of cyclodextrins in a fluidized-bed reactor using cyclodextrin-glycosyl-transferase

Cyclodextrin-glycosyl-transferase (EC2.4.1.19), produced by Wacker (Munich, Germany), was purified by biospecific affinity chromatography with β-cyclodextrin (β-CD) as ligand, and immobilized into controlled pore silica particles (0.42 mm). This immobilized enzyme (IE) had 4.7 mg of protein/g of support and a specific activity of 8.6 μmol of β-CD/(min·g_IF) at 50°C, pH 8.0. It was used in a fluidized-bed reactor (FBR) at the same conditions for producing cyclodextrins (CDs) with 10% (w/v) maltodextrin solution as substrate. Bed expansion was modeled by the Richardson and Zaki equation, giving a good fit in two distin ctranges of bed porosities. The minimum fluidization velocity was 0.045 cm/s, the bed expansion coefficient was 3.98, and the particle terminal velocity was 2.4 cm/s. The FBR achieved high productivity, reaching in only 4 min of residence time the same amount of CDs normally achieved in a batch reactor with free enzyme after 24h of reaction, namely, 10.4 mM β-CD and 2.3 mM γ-CD.     

Application of combined thermoanalytical techniques in the investigation of cyclodextrin inclusion complexes

Citronellol and citronellyl acetate have been entrapped with α-, β- and γ-cyclodextrin (CD). Evolved gas detection and TG-MS coupling was applied to prove the actual inclusion complex formation between monoterpens and CDs. The terpene content was determined by UV-VIS specrophotometry and RP-HPLC and the effect of storage time on the terpene content was also investigated. The α- and γ-cyclodextrin inclusion complexes showed higher thermal stabilities vs. dynamic heating compared to the β-CD complexes. On the contray, the retention of guest using β-cyclodextrin even after 10 years of storage was much more pronounced. Experimental data other than 1:1 complex compositions are assumed. Molecular modeling experiments also suggested multiple complex compositions.     

Interactions of tetrakis(4-N-methylpyridyl)porphyrin with cyclodextrins and disodium phthalate in aqueous solution

In pH 7.3 buffers, the interactions of a cationic porphyrin, tetrakis(4-N-methylpyridyl)porphyrin (TMPyP), with cyclodextrins (CDs) and disodium phthalate (DSP) have been examined by means of absorption, fluorescence, and induced circular dichroism spectroscopy. α-CD, β-CD, and γ-CD form a 1:1 inclusion complex with a TMPyP monomer, which dimerizes in solution without CD. TMPyP also forms a 1:1 organic cation–organic anion complex with DSP. The 1:1 TMPyP–DSP complex forms a ternary CD–TMPyP–DSP inclusion complex with α-, β-, and γ-CD, in which a DSP molecule is not incorporated into the CD cavity. From the fluorescence intensity change, the␣equilibrium constants have been evaluated for the formation of the inclusion complexes and the organic cation–anion complexes.     

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.     

Thermoanalytical Study of the Dehydration of Cyclodextrin Inclusion Complexes of Clofibric Acid

Hydrated inclusion complexes of the hosts β-CD (CD=cyclodextrin), γ-CD and permethylated β-CD with the guest clofibric acid were analysed by TG and DSC methods to characterise their dehydration behaviours. Activation energies for dehydration of the β- and γ-CD clofibric acid complexes, determined by isothermal thermogravimetry, are significantly lower (∼20-25%) than those for the corresponding uncomplexed hydrated CDs. These data can be reconciled with X-ray structural data which show that H₂O molecules in the complexes occupy different crystal sites from those occupied in the parent CDs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Binary Systems of Nifedipine And Various Cyclodextrins in The Solid State. Thermal,FTIR, XRD studies

Nifedipine complexes with β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), 2-hydroxypropyl-β-cyclodextrin (2HP-β-CD), randomly methylated-β-cyclodextrin (RM-β-CD) and heptakis(2,6-O-dimethyl)-β-cyclodextrin (DM-β-CD) have been prepared by both kneading and heating methods and their behaviour studied by differential scanning calorimetry (DSC), diffuse reflectance mid-infrared spectroscopy (FTIR) and X-ray diffractometry (XRD). DSC revealed the nifedipine melting endotherm with onset at approximately 171°C for the kneaded mixtures with β-CD, γ-CD and 2HP-β-CD, thus confirming the presence of nifedipine in the crystalline state, while some decrease in crystallinity was observed in the DM-β-CD kneaded mixture. With RM-β-CD, however, broadening and shifting of the nifedipine endotherm and reduction in its intensity suggested that the kneading could have produced an amorphous inclusion complex. These differing extents of interaction of nifedipine with the cyclodextrins were confirmed by FTIR and XRD studies. This revised version was published online in August 2006 with corrections to the Cover Date.     

Separation of the enantiomers of four chiral drugs by neutral cyclodextrin-mediated capillary zone electrophoresis

Summary Neutral cyclodextrin (CD)-modified capillary zone electrophoresis (CZE) has been applied to the chiral separation of four basic drugs— clorprenaline, benzhexol, esmolol and terazosin. Selector screening and concentration optimization experiments were performed. The resolution was 3.9 for clorprenaline, 2.3 for benzhexol, 3.1 for esmolol and 1.2 for terazosin when the running electrolyte was 60 mM hydroxypropyl-β-CD, 15 mM heptakis (2,3,6-Tri-O-methyl)-β-CD, 60 mM γ-CD and 60 mM heptakis (2,6-di-O-methyl)-β-CD, respectively, in 50 mM, pH 2.5 sodium phosphate buffer.     

A new example of reversal of the order of migration of enantiomers, as a function of cyclodextrin concentration and pH, by cyclodextrin-modified capillary zone electrophoresis: enantioseparation of 6,6′-dibromo-1,1′-binaphthyl-2,2′-diol

Enantioseparation of 6,6′-dibromo-1,1′-binaphthyl-2,2′-diol (DBBD) by cyclodextrin-modified capillary zone electrophoresis (CD-CZE) was studied using the three native α, β, and γ cyclodextrins, the three hydroxypropylated cyclodextrins (2-hydroxypropyl-α, β, and γ), heptakis-2,6-di-O-methyl-β-CD (DM-β-CD), and heptakis-2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD). First, the acidity constants of DBBD were determined using capillary electrophoresis, before performing enantioseparation. The influence of the concentrations of the studied cyclodextrins on the enantioseparation was explored and the experimental optimal concentrations were determined and compared to the theoretical optimal concentrations. Moreover, the apparent complexation constants between each studied cyclodextrin and the two DBBD enantiomers were evaluated using a non-linear curve fitting method and three linear plotting methods (x-reciprocal, y-reciprocal and double reciprocal). For TM-β-CD, the order of migration of the enantiomers of DBBD reversed as a function of TM-β-CD concentration. The influence of the nature of methylated cyclodextrin derivatives (methyl-β-CD (M-β-CD) and DM-β-CD) was then studied. Inversion of the order of migration of the enantiomers of DBBD was observed for DM-β-CD, whereas the S enantiomer of DBBD always migrated first for M-β-CD.