Highly Soluble β-Cyclodextrin Derivatives

β-Cyclodextrin is an inclusion complexing agent produced on industrial scale. The low solubility of its complexes is a serious disadvantage in many cases. Substituted β-Cyclodextrin derivatives are characterized by much higher solubility. Among methylated derivatives the heptakis-di-O-methyl-β-Cyclodextrin deserves special attention. In its aqueous solutions many poorly soluble or practically insoluble compounds can be dissolved, whereas the solubility enhancement factor is between 3 and 1 200 depending on the substance. Aqueous injectable solutions can be prepared from highly lipophilic compounds, enzymatic reaction rate of lipophilic substrates can be strongly accelerated, biological absorption of drugs or fat-soluble vitamins can be enhanced and vaccine production can be augmented up to 100-fold. Methylated cyclodextrins are able to act as solvents by forming watersoluble inclusion complexes and as phase-transfer catalysts at free radical polymerization. Low molecular cyclodextrin-polymers with molecular weight between 2 000 and 10 000 behave similarly. They are very well soluble, retaining their complexing ability. On account of the macromolecular character the diffusion of dissolved and complexed substances is considerably decelerated. By including into a chiral cavity the entrapped molecules become optically active oriented, and therefore their reactions are sterically controlled.     

Morphological Study of Products Containing β-Cyclodextrin

β-Cyclodextrin (β-CD) has been applied in various fields of drug formulation. Its presence in powder Paracetamol (PAR) products prepared with different methods (kneading and spray drying) changes the morphology of original drug. The product made by spray drying has nearly isodimensional spherical particles, and potentially better flowability than the kneaded product. Spray drying of PAR product without β-CD shows similar morphology like a product with β-CD, but the particles show a greater inclination to adhesion. The results were obtained by electron microscopic study.     

Electron Microscopic Study of Products Containing β-Cyclodextrin

The advantageous properties of cyclodextrin (CD) derivatives have been applied in various fields of drug production. Their presence in physical powder mixtures increases the flow characteristics, which can be improved still further if isodimensional particles and homogeneous dispersive products are produced by spray-embedding. An account is given of an electron microscopic study of such products.     

Oxidative Degradation of β-Cyclodextrin Induced by Lipid Peroxidation

The oxidative degradation of β-cyclodextrin (β-CD) induced by autoxidation of linoleate has been investigated in the solid system composed of β-CD and linoleate. β-CD was oxidized with a propagative oxidation of linoleate to induce the cleavage of its glucosidic linkage and this degradation proceeded proportionally with the moisture content in the solid system. The oxidative cleavage of β-CD gave several kinds of oligosaccharides which were composed of D -erythrose, D -arabinose, D -erythropentosulose, D -xylopentdial-dose, D -glucose and deoxyunsaturated hexose as their reducing terminals. These degradation of β-CD seemed to be initiated by certain radical species formed from the peroxidation of linoleate.     

Improved Reaction Conditions for Preparation of β-Cyclodextrin (β-CD) from Cassava (Manihot esculenta Crantz) Starch

Experimental conditions for bioconversion of starch to get higher yield of ß-cyclodextrin using cyclodextrin glycosyl transferase (CGT-ase) (EC 2.4.1.19) were worked out. A 5% initial starch concentration gave ß-CD yield of 20-28% that could be separated as ß-CD bromobenzene complex from the reaction mixture. Addition of bromobenzene during the second phase of the reaction, i.e. CD formation phase, was found beneficial.     

Purification and physicochemical characterization of ovine β‐lactoglobulin and α‐lactalbumin

Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion‐exchange chromatography and reversed‐phase high‐performance liquid chromatography (HPLC) showed the presence of two fractions of β‐lactoglobulin but only one of α‐lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation‐mass spectrometry (ESI‐MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine β‐lactoglobulin. Amino acid compositions of the two variants of β‐lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of β‐lactoglobulin A compared to β‐lactoglobulin B at pH 2, and indicated high instability of ovine α‐lactalbumin at this pH.     

Halogen Azide Displacement to Prepare Some Symmetrically Substituted β-Cyclodextrin Derivatives

The heptakis (2,3-di-O-acetyl-6-bromo-6-deoxy)-β-cyclodextrin has been converted to the 6-azido derivate with 82% yield, then deacetylated by the Zemplén's method (yield 75%) and methylated by methyl-iodide (yield 66%), to heptakis (2,3-di-O-methyl-6-azido-6-deoxy)-β-cyclodextrin, which is an appropriate intermediary toward methylated amino-CD-derivatives. The structure of all compounds were unambiguously proven by NMR spectroscopy.     

Optimization of Cultivation Conditions for the Production of γ-Cyclodextrin Glucanotransferase by Bacillus macorous

The production of γ-cyclodextrin glucanotransferase (γ-CGTase) from Bacillus macorous WSH02-06 was optimized in shake flasks using conventional sequential techniques and statistical experimental design. Effects of nutrients including carbon and nitrogen sources, cation ions, initial pH, and temperature on γ-CGTase production were investigated. Corn starch, peptone, Mn²⁺, and Zn²⁺ were found to be essential for obtaining high γ-CGTase activity and biomass. The promoting effect of manganese and zinc on enzyme production has not been reported previously. According to the results of orthogonal array experiment, the optimal culture medium for high γ-CGTase activity was determined. Maximal γ-CGTase activity obtained in the optimized culture broth was about 250 U/mL which was 10-fold higher than that obtained in the basal medium. Time course of cell growth and γ-CGTase production in a 7 l fermenter showed that enzyme production was growth associated, and that maximum γ-CGTase activity reached 277 U/mL in 17 h.     

The Action of Bacillus subtilis Saccharifying Amylase on Starch and β-Cyclodextrin

The action of B. subtilis saccharifying amylase on starch has been examined. It has been found that high yields of reducing sugar and glucose are produced from both starch solutions and starch gels upon prolonged reaction. The enzyme has also been found to have maltase and cyclodextrinase activities and to produce an almost quantitative yield of glucose from β-cyclodextrin. Attempts to separate or differentiate the amylase, maltase and cyclodextrinase activities by electrophoresis, inhibitors, heat inactivation and hydroxylapatite chromatography have been unsuccessful, and it is concluded that the enzyme activities are all due to one molecular species.     

Die Wirkung von α-Amylasen auf β-Cyclodextrin und der Nachweis von α-Amylasefremdaktivitäten in ausgewählten Enzympräparaten

The action of α-amylases on β-cyclodextrin and the evidence of foreign activity of α-amylase in selected preparations of enzymes The interaction between cyclodextrins and α-amylases taken from different sources is discribed contradictious in the literature. Some α-amylases e.g. isolated from Aspergillus oryzae, porcine pancreas and saliva hydrolized cyclodextrins to glucose. The hydrolysis of cyclodextrins catalysed by α-amylase from Bacillus species have been described conflicting. In this paper the action of hydrolysis of different preparations of α-amylases on β-cyclodextrin have been investigated. It has been shown that Rohalase M3 (α-amylase from Aspergillus niger) cleaves the ring of β-cyclodextrin. 2 α-amylases from Bacillus subtilis are not able to hydrolyse β-cyclodextrin. The reasons for the different actions of hydrolysis have been discussed with size and structure of the active centre of the enzymes. Moreover, different preparation of hydrolysis have been tested on secondary activity of α-amylase. 2 glucoamylases from Aspergillus oryzae have been shown secondary activity of α-amylase. With the hydrolases α-glucosidase from fungies, β-amylase from malt, saccharase from yeast, invertase from S. cerevisiae and pullulanase from Aerobacter aerogenes no cleavage of the ring of β-cyclodextrin could be detected.     

Purification of properties of Saccharomyces cerevisiae cystathionine β-synthase

Cystathionine β-synthase (β-CTSase), which catalyses cystathionine synthesis from serine and homocysteine, was purified to homogeneity from Saccharomyces cerevisiae. The molecular mass of the enzyme was estimated to be 235 kDa by gel filtration and 55 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, indicating that it is a homotetramer. The N-terminal amino acid sequence of the enzyme perfectly coincided with that deduced from the nucleotide sequence of CYS4, except for the absence of initiation methionine. The purified β-CTSase catalysed cysteine synthesis from serine (or O-acetylserine) and H₂S. From this finding, we discuss the multifunctional nature and evolutionary divergence of S-metabolizing enzymes.     

β-GLUCAN AND β-GLUCANASE IN BREWING

The development of endo β-glucanase during the micro-malting of barley, with and without the addition of gibberellic acid, was compared. Results indicated that the stimulative effect of gibberellic acid on the enzyme system was of only marginal practical importance. From the assessment of the enzyme activity in a number of commercial malts it would appear that the germination time used for some malts is too short to take full advantage of the critical phase of very rapid enzyme development. The viscosity-reducing power of β-glucanase was demonstrated in miniature brewery mashing experiments and details of the progressive degradation of β-glucan by the enzyme system were analysed by gel filtration methods. The β-glucan content of a number of varieties of barley was established.     

Study on Interactions of Corn Starch and γ-Cyclodextrin with Lipid Peroxide

The deterioration of corn starch and γ-cyclodextrin (γ-CD) as a model compound by coexisting of methyl linolate (ML) has been studied in the solid system. With the elapse of oxidation time of ML, the reducing values of above the sugars increased and the value of γ-CD with Silica Gel done more largely than without the one. The molecular weight of corn starch may decrease slightly, the glycosidic linkages of amylopectin and amylose molecules in the starch may be cleaved partially and the crystalline structure of the starch will be deteriorted a little by some active radicals formed by the autoxidation of ML. The propagative autoxidation of ML induced the deterioration of γ-CD giving the two processes of the hydrolysis and the depolymerization in the similar way as observed in the reaction of γ-CD with OH radicals.     

Purification of α-D-Glucose-1-phosphate

A method is described for the purification of α-D-glucose-1-phosphate. The compound can be isolated from reaction mixtures containing carbohydrate, inorganic phosphate and glucose-1-phosphate by a one step chromatographic proces using an anion-exchange resin with potassiumacetate as eluting buffer. Glucose-1-phosphate can than be precipitated directly from the potassiumacetate solution by the addition of ethanol, because potassiumacetate is very soluble in ethanol and ethanol-water mixtures.     

Purification and Characterization of a β-Amylase of Hendersonula toruloidea

β-Amylase produced by Hendersonula toruloidea was purified to homogeneity by salting out with ammonium sulphate, ion-exchange chromatography on DEAE-cellulose and gel-filtration on Sephadex G-75. The relative molecular mass of the enzyme was estimated to be 60,000 by gel filtration. The enzyme was optimally active at pH 6.0 and 60°C, stable between pH 6 and 8 (24 h) and retained 74% activity at 70°C (30 min). It was strongly activated by Na⁺ but inhibited by Hg²⁺, Zn²⁺ and Cu²⁺. The enzyme hydrolyzed amylopectin (Km 0.42 mg/ml) forming maltose, maltotetraose and unidentified maltooligosaccharide, and hydrolyzed soluble starch (Km 0.3 mg/ml) and glycogen (Km 0.5 mg/ml) forming maltose and unidentified maltooligosaccharide.     

Purification of an Arabinofuranosidase and Two β‐Xylopyranosidases from Germinated Wheat

Arabinosidase and β‐xylosidase activities were detected in germinated wheat grain, and both increased over seven days of germination, under malting conditions. Arabinosidase was partially purified by anion exchange chromatography, chromatofocusing and gel filtration chromatography. The pH optimum of the partially purified enzyme was 4.2 and the K_M was 1.90 mM p‐NP‐Ara. Edman degradation, MALDI‐TOF mass spectrometry and nano‐ESI mass spectrometry were used to identify the two major proteins in the partially purified arabinosidase mixture. The two proteins were a β‐amylase with an amino acid sequence partially homologous to a barley β‐amylase, and three wheat serine protease inhibitors. Further purification, by affinity chromatography and hydrophobic interaction chromatography, removed the identified contaminating proteins. At this point an 80 kDa protein was detected by SDS‐PAGE. No identity could be assigned to this protein by MALDI‐TOF mass spectrometry by reference to electronic protein databases. Similarly, the β‐xylosidase was partially purified by anion exchange chromatography followed by chromatofocusing and gel filtration chromatography. The first step separated the mixture into two distinct fractions with K_M values of 3.35 and 4.01 mM p‐NP‐Xyl and pH optima of 4.5. The latter fraction also displayed xylanase activity against RBB‐xylan.     

Synthesis of β‐Galactooligosaccharides from Lactose Using Microbial β‐Galactosidases

Abstract: Galactooligosaccharides (GOSs) are nondigestible oligosaccharides and are comprised of 2 to 20 molecules of galactose and 1 molecule of glucose. They are recognized as important prebiotics for their stimulation of the proliferation of intestinal lactic acid bacteria and bifidobacteria. Therefore, they beneficially affect the host by selectively stimulating the growth and/or activity of a limited number of gastrointestinal microbes (probiotics) that confer health benefits. Prebiotics and probiotics have only recently been recognized as contributors to human health. A GOS can be produced by a series of enzymatic reactions catalyzed by β‐galactosidase, where the glycosyl group of one or more D‐galactosyl units is transferred onto the D‐galactose moiety of lactose, in a process known as transgalactosylation. Microbes can be used as a source for the β‐galactosidase enzyme or as agents to produce GOS molecules. Commercial β‐galactosidase enzymes also do have a great potential for their use in GOS synthesis. These transgalactosyl reactions, which could find useful application in the dairy as well as the larger food industry, have not been fully exploited. A better understanding of the enzyme reaction as well as improved analytical techniques for GOS measurements are important in achieving this worthwhile objective.