Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with gamma-cyclodextrin

Cyclodextrin glycosyltransferase catalyzes the formation of a mixture of cyclodextrins from starch by an intramolecular transglycosylation reaction. To manipulate the product specificity of the Paenibacillus sp. A11 and Bacillus macerans cyclodextrin glycosyltransferases towards the preferential formation of gamma-cyclodextrin (CD(8)), crosslinked imprinted proteins of both cyclodextrin glycosyltransferases were prepared by applying enzyme imprinting and immobilization methodologies. The crosslinked imprinted cyclodextrin glycosyltransferases obtained by imprinting with CD(8) showed pH and temperature optima similar to those of the native and immobilized cyclodextrin glycosyltransferases. However, the pH and temperature stability of the immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were higher than those of the native cyclodextrin glycosyltransferases. When the catalytic activities of the native, immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were compared, the efficiency of the crosslinked imprinted enzymes for CD(8) synthesis was increased 10-fold, whereas that for cyclodextrin hydrolysis was decreased. Comparison of the product ratios by high-performance anion exchange chromatography showed that the native cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced CD(6) : CD(7) : CD(8) : > or = CD(9) ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0 after 24 h of reaction at 40 degrees C with starch substrates. In contrast, the crosslinked imprinted cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced cyclodextrin in ratios of 15 : 20 : 50 : 15 and 17 : 14 : 49 : 20, respectively. The size of the synthesis products formed by the crosslinked imprinted cyclodextrin glycosyltransferases was shifted towards CD(8) and > or = CD(9), and the overall cyclodextrin yield was increased by 12% compared to the native enzymes. The crosslinked imprinted cyclodextrin glycosyltransferases also showed higher stability in organic solvents, retaining 85% of their initial activity after five cycles of synthesis reactions.     

Review: cyclodextrins and their interaction with amylolytic enzymes

This review is concerned with inhibition of amylases by cyclodextrins (cyclic maltooligosaccharides), the interaction that occurs between amylases and cyclodextrins and the application of cyclodextrin affinity chromatography in the purification of amylases. In many cases, amylases that are competitively inhibited by cyclodextrins can be purified by cyclodextrin affinity chromatography with the cyclodextrins interacting with the active site on such enzymes. Interestingly amylases that are not competitively inhibited by cyclodextrins may also be purified by cyclodextrin affinity chromatography. Therefore, cyclodextrin affinity chromatography can function in the purification of such amylolytic enzymes with the interaction occurring at a site removed from the active site. In such cases it appears that the cyclodextrin is interacting with an affinity site or binding site that is present on some amylolytic enzymes. It seems that certain similarities occur among the binding sites of such enzymes. Literature concerning amylases, and their subsequent purification using cyclodextrin affinity chromatography is reviewed and the fundamental basis of the interaction of the cyclodextrin with amylolytic enzymes is discussed here.     

Use of cyclodextrin as an agent to induce excretion of Bordetella pertussis antigens

Abstract This paper attempts to provide an explanation for the effect of cyclodextrin on the yield of Bordetella pertussis soluble antigens. It was demonstrated that the addition of cyclodextrin to the synthetic Stainer-Scholte liquid medium enhances the level of the intracellular form of adenylate cyclase (200 kDa) in the supernate. In addition to this effect, it has been reported that cyclodextrin also enhances the levels of two other extracellular proteins, pertussis toxin and filamentous hemagglutinin. As these antigens are structurally different, it seems that the effect of cyclodextrin is not specific. With the use of different buffer systems of well-known action on outer membrane stability it was possible to determine a relationship between the presence of cyclodextrin, destabilisation of the outer membrane and the release of proteins. It was determined that the cyclodextrin did not modify the fluidity of B. pertussis cells but produced a change of outer membrane permeability.     

Chemical modification of lysine side chains of cyclodextrin glycosyltransferase from Thermoanaerobacter causes a shift from cyclodextrin glycosyltransferase to alpha-amylase specificity

Cyclodextrin glycosyltransferases and alpha-amylases are two groups of enzymes with related secondary structures. However, cyclodextrin glycosyltransferases display transferase activities not present in alpha-amylases, probably derived from the existence of two more domains and different amino acid sequences. The hydrolytic activity of cyclodextrin glycosyltransferases is generally quite low, except for two cyclodextrin glycosyltransferases from termophiles. In this work, we have carried out the chemical modification (with acetic anhydride) of the amino groups of cyclodextrin glycosyltransferase from Thermoanaerobacter to assess their contributions to protein function. The acetylated cyclodextrin glycosyltransferase showed a significant reduction of its cyclization, coupling and disproportionation activities. Surprisingly, the hydrolytic (saccharifying) activity was slightly enhanced. These results suggest the participation of one or more lysine side chains in the interactions contributing to the transferase activity, either in any of the S11 subsites or in the acceptor binding site.     

Studies on enzymatic continuous production of cyclodextrins in an ultrafiltration membrane bioreactor

Studies on simultaneous hydrolysis of starch and synthesis of cyclodextrins by Thermo-aerobacter cyclodextrin glucosyltransferase were conducted in an ultrafiltration membrane bioreactor, allowing enzyme recovery and reduction of product inhibition. The influence of various reaction parameters like starch concentration, enzyme dosage and residence time on cyclodextrin composition was tested. A comparison of batch and continuous cyclodextrin production indicates that employing an ultrafiltration membrane bioreactor increases process efficiency.     

Response Surface Methodology to Optimize Novel Fast Disintegrating Tablets Using β Cyclodextrin as Diluent

The objective of this work was to apply response surface approach to investigate main and interaction effects of formulation parameters in optimizing novel fast disintegrating tablet formulation using β cyclodextrin as a diluent. The variables studied were diluent (β cyclodextrin, X ₁), superdisintegrant (Croscarmellose sodium, X ₂), and direct compression aid (Spray dried lactose, X ₃). Tablets were prepared by direct compression method on B2 rotary tablet press using flat plain-face punches and characterized for weight variation, thickness, disintegration time (Y ₁), and hardness (Y ₂). Disintegration time was strongly affected by quadratic terms of β cyclodextrin, croscarmellose sodium, and spray-dried lactose. The positive value of regression coefficient for β cyclodextrin suggested that hardness increased with increased amount of β cyclodextrin. In general, disintegration of tablets has been reported to slow down with increase in hardness. However in the present study, higher concentration of β cyclodextrin was found to improve tablet hardness without increasing the disintegration time. Thus, β cyclodextrin is proposed as a suitable diluent to achieve fast disintegrating tablets with sufficient hardness. Good correlation between the predicted values and experimental data of the optimized formulation validated prognostic ability of response surface methodology in optimizing fast disintegrating tablets using β cyclodextrin as a diluent.     

The cyclization mechanism of cyclodextrin glycosyltransferase (CGTase) as revealed by a gamma-cyclodextrin-CGTase complex at 1.8-A resolution

The enzyme cyclodextrin glycosyltransferase is closely related to alpha-amylases but has the unique ability to produce cyclodextrins (circular alpha(1-->4)-linked glucoses) from starch. To characterize this specificity we determined a 1.8-A structure of an E257Q/D229N mutant cyclodextrin glycosyltransferase in complex with its product gamma-cyclodextrin, which reveals for the first time how cyclodextrin is competently bound. Across subsites -2, -1, and +1, the cyclodextrin ring binds in a twisted mode similar to linear sugars, giving rise to deformation of its circular symmetry. At subsites -3 and +2, the cyclodextrin binds in a manner different from linear sugars. Sequence comparisons and site-directed mutagenesis experiments support the conclusion that subsites -3 and +2 confer the cyclization activity in addition to subsite -6 and Tyr-195. On this basis, a role of the individual residues during the cyclization reaction cycle is proposed.     

Effect of different substrates and their preliminary treatment on cyclodextrin production

Summary Various kinds of substrates were tested for cyclodextrin production with cyclodextrin glucanotransferase (CGTase) from Bacillus megaterium. The enzyme formed cyclodextrin from different kinds of starch, dextrins, amylose, and amylopectin. However, the highest degree of conversion was obtained from starch. Corn starch appeared to be the best substrate – the cyclodextrin yield was 50.9%. The effect of molecular mass and preliminary treatment of starch with α-amylase on the CD yield was investigated. It was proved that CGTase preferred native starch with high molecular mass and low dextrose equivalent. The preliminary treatment with α-amylase occurred to be inefficient and unnecessary since it did not lead to an increase in the CD yield. Some of the substrates were treated with pullulanase. The effect of debranching was highest in the case of corn starch: the cyclodextrin yield increased by 10%.     

Comparison of three S‐β‐CDs with different degrees of substitution for the chiral separation of 12 drugs in capillary electrophoresis

Three kinds of sulfated β‐cyclodextrin (S‐β‐CD), including a single isomer, heptakis‐6‐sulfato‐β‐cyclodextrin (HS‐β‐CD), degree of substitution (DS) of 7, which was synthesized in our laboratory and another two commercialized randomly substituted mixtures, a sulfated β‐cyclodextrin with DS of 7 to 11, as well as a highly sulfated‐β‐cyclodextrin with DS of 12 to 15, were used for the enantioresolution of 12 drugs (the β‐blockers, phenethylamines, and anticholinergic agents) in capillary electrophoresis. The enantioseparation under varying concentrations of S‐β‐CD and background electrolyte pH were systematically investigated and compared. Based on the experimental results, the effect of the nature of S‐β‐CD and analyte structure on the enantioseparation is discussed.     

The role of membrane stiffness and actin turnover on the force exerted by DRG lamellipodia

We used optical tweezers to analyze the effect of jasplakinolide and cyclodextrin on the force exerted by lamellipodia from developing growth cones (GCs) of isolated dorsal root ganglia (DRG) neurons. We found that 25 nM of jasplakinolide, which is known to inhibit actin filament turnover, reduced both the maximal exerted force and maximal velocity during lamellipodia leading-edge protrusion. By using atomic force microscopy, we verified that cyclodextrin, which is known to remove cholesterol from membranes, decreased the membrane stiffness of DRG neurons. Lamellipodia treated with 2.5 mM of cyclodextrin exerted a larger force, and their leading edge could advance with a higher velocity. Neither jasplakinolide nor cyclodextrin affected force or velocity during lamellipodia retraction. The amplitude and frequency of elementary jumps underlying force generation were reduced by jasplakinolide but not by cyclodextrin. The action of both drugs at the used concentration was fully reversible. These results support the notion that membrane stiffness provides a selective pressure that shapes force generation, and confirm the pivotal role of actin turnover during protrusion.     

Synthesis of Maltosyl(α1→6)cyclodextrins through the Reverse Reaction of Thermostable Bacillus acidopullulyticus Pullulanase

Maltosyl(α1→6)α-, β or γ-cyclodextrin was synthesized from maltose and α-, β- or γ- cyclodextrin, respectively, using Bacillus acidopullulyticus pullulanase (EC 3.2.1.41). More than 40% of each cyclodextrin substrate was converted to the corresponding maltosyl(α1→6)cyclodextrin under the conditions given below; the combined concentration of maltose and cyclodextrin was 70 ~ 75 % (w/w), the molar ratio of maltose to cyclodextrin was 9~18, and the amount of pullulanase was 100~200units/g of cyclodextrin. The optimum pH and temperature for the formation of maltosyl(α1→6)cyclodextrins were 4.0—4.5 and 60~70°C, respectively. Each maltosyl(α1→6)-cyclodextrin produced was separated from noncyclic saccharides, maltose and branched tetraose, by methanol and ethanol precipitations. The maltosyl(α1→6)cyclodextrins were further purified by gel filtration on a Toyopearl HW 40 S column and crystallization from aqueous (for maltosyl(α1→6)β-cyclodextrin) or methanol (for maltosyl(α1→6)β-cyclodextrin) solution. From 10 g each of the corresponding cyclodextrin, the yields of the purified maltosyl(α1→6)α-, β- and γ-cylcodextrins were 3.0 ~ 3.6 g, 2.5 ~ 2.8g and 2.2 ~ 2.5 g, respectively. Identification of the maltosyl(α1-6)cyclo-dextrins was performed by means of hydrolysis with Klebsiella pneumoniae pullulanase, methyla- tion analysis and ¹³C-NMR analysis.     

A new fluorescent chemosensor for cadmium(II) based on a pyrene‐appended piperidone derivative and its β‐cyclodextrin complex

We report, in this article, a piperidin‐4‐one derivative carrying pyrenyl fluorescent reporter groups which acts as a Cd²⁺ ion sensor. The compound is synthesized and characterized using IR and NMR spectral techniques. The compound forms an inclusion complex with β‐cyclodextrin. It selectively binds to Cd²⁺ ions in water and aqueous β‐cyclodextrin media. The stoichiometry of the host–guest complex of the compound with β‐cyclodextrin is 1:2. The ligand–metal ion binding stoichiometry is 1:1 both in water and in β‐cyclodextrin. The linear concentration range of detection of the metal ion is reported. Cyclodextrin complex formation does not affect the metal ion selectivity of the compound.     

Guest-induced conformational change of β-cyclodextrin capped with an environmentally sensitive chromophore

A capped cyclodextrin, 6-deoxy-6-(p-hydroxy-m-nitrophenacylthio)-β-cyclodextrin, was prepared in order to detect any conformational change of the host upon the guest binding. The association constant between the cyclodextrin and 1-adamantanecarboxylate, cyclohexanecarboxylate, p-methylbenzoate, 3,3-dimethylbutyrate, or 2,2-dimethylpropionate was enhanced 20, 5.3, 3.7, 2.3, or 2.0 times, respectively, by chromophore capping. The changes in the electronic, NMR, and circular dichroism spectra as well as pK_a of this cyclodextrin upon binding of the guest strongly indicate a conformational change around the chromophoric moiety of the cyclodextrin.     

Engineering of cyclodextrin glucanotransferase on the cell surface of Saccharomyces cerevisiae for improved cyclodextrin production

The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.     

Carboxymethylated cyclodextrins and their complexes with Pr(III) and Yb(III) as water‐soluble chiral NMR solvating agents for cationic compounds

Cyclodextrins that are indiscriminately carboxymethylated at the 2‐, 3‐, and 6‐positions are used as chiral NMR solvating agents for cationic substrates with phenyl, naphthyl, pyridyl, indoline, and indole rings. Enantiodifferentiation with the α‐, β‐, and γ‐cyclodextrin derivatives is compared. The carboxymethylated derivatives are almost always more effective as chiral NMR solvating agents for cationic substrates than native cyclodextrins. The most effective carboxymethylated cyclodextrin varies for different substrates, and at times even different resonances of the substrate. Addition of paramagnetic praseodymium(III) or ytterbium(III) to mixtures of the carboxymethylated cyclodextrin and substrate often causes enhancements in enantiomeric discrimination and facilitates measurements of enantiomeric purity. The lanthanide ion bonds to the carboxymethyl groups and causes perturbations in the chemical shifts in the NMR spectra of substrate molecules in the cyclodextrin cavity. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

环糊精葡萄糖基转移酶高效异源表达研究进展

环糊精葡萄糖基转移酶(cyclodextringlycosyltransferase,CGTase)酶法合成环糊精是目前生产环糊精的主要方法。本文介绍了用于生产环糊精葡萄糖基转移酶的几种工程菌株:大肠杆菌、枯草芽孢杆菌以及毕赤酵母,其中大肠杆菌是目前应用最广泛的用于表达CGTase的表达系统。除此之外,本文还总结了高效表达环糊精葡萄糖基转移酶的有效策略:选择合适的表达载体、启动子以及信号肽,以及密码子优化和分子伴侣共表达,以期为在相关CGTase研究领域开展研究提供参考。     

Evidence for covalent binding between copper ions and cyclodextrin cavity: a vibrational circular dichroism study

Vibrational absorption and circular dichroism (VCD) spectra were obtained for parent cyclodextrins, hydroxyl deuterated alpha-cyclodextrin, cyclodextrin-copper complexes, and for the cyclodextrin inclusion complexes with Methyl Orange, methyloxirane, 1-propanol, and substituted cyclohexanones, in the solution phase. Changes in the VCD spectra, reflecting perturbations of cyclodextrin cavity, were found in the case of an inclusion complex with Methyl Orange, but for the remaining inclusion complexes measurable changes in VCD were not found. Significant changes observed in the VCD spectra of cyclodextrin-copper complexes suggest that the covalent binding of copper ions to the hydroxyl groups of cyclodextrin is involved.     

Molecular mass control using polyanionic cyclodextrin derivatives for the epsilon-poly-l-lysine biosynthesis by Streptomyces

To control the molecular mass of a natural polycationic, antimicrobial, Streptomyces-biosynthesized polymer, epsilon-poly-l-lysine, addition of polyanionic cyclodextrin derivatives to the culture medium was evaluated. Chemically modified cyclodextrins such as a sulfated cyclodextrin caused a notable shortening of the polymer length of epsilon-poly-l-lysine from 3.5 to 4.5 kDa to less than 2.5 kDa by the enforcing action of glycerol, which has a weak potential to inhibit polymer elongation by terminal esterification. Meanwhile, polyanionic cyclodextrin inhibited the shortening action with n-octanol, which has a strong ability to inhibit polymer elongation through an undetermined function. The design of chemical structures of polyanionic cyclodextrin, optimization of their addition concentrations, and selection of hydroxyl compounds coexisting with them are critical for this simple and easy method for polymer length control and terminal modification of epsilon-poly-l-lysine.     

Specific alpha-cyclodextrin production by a novel thermostable cyclodextrin glycosyltransferase

Summary A novel bacterial isolate, Bacillus amyloliquefaciens strain AL 35, produced high yields of a cyclodextrin glycosyltransferase (CGT'ase) when grown in a submerged culture. The stability of CGT'ase to high temperature and alkaline pH enabled processing for cyclodextrin production to be carried out at 60° C and pH 9.0. Crude culture filtrates containing the CGT'ase could convert gelatinized starch substrates to predominatly -cyclodextrins (up to 95% of the total cyclodextrin yields).     

Engineering of Cyclodextrin Glucanotransferase on the Cell Surface of Saccharomyces cerevisiae for Improved Cyclodextrin Production

The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.