Seçil Önal

Inhibition of alpha-glucosidase by aqueous extracts of some potent antidiabetic medicinal herbs.

Abstract Diabetes mellitus is one of the most prevalant diseases of adults. Agents with α‐glucosidase inhibitory activity have been useful as oral hypoglycemic drugs for the control of hyperglycemia in patients with type 2; noninsulin‐dependent, diabetes mellitus (NIDDM). Investigation of some medicinal herbs: Urtica dioica, Taraxacum officinale, Viscum album, and Myrtus communis with α‐glucosidase inhibitor activity was conducted to identify a prophylactic effect for diabetes in vitro. All plants showed differing potent α‐glucosidase inhibitory activity. However, Myrtus communis strongly inhibited the enzyme (IC50 = 38 µg/mL). The inhibitory effect of these plants and some common antidiabetic drugs against the enzyme source (bakers yeast, rabbit liver, and small intestine) were also searched. Approximately all inhibitors used in this study showed quite different inhibitory activities, according to α‐glucosidase origins. Furthermore, subsequent separation of the active material from Myrtus communis by HPLC showed that only one fraction acted as an α‐glucosidase inhibitor.

Molecularly imprinted polymers for separation of various sugars from human urine.

Molecularly imprinted polymers were the new, simple and unexpensive materials that can be used in several clinical applications. Phenylboronic acid has been frequently used as functional monomer for the covalent imprinting of diols. In this study, the phenylboronic acid esters of fructose, galactose, glucose and raffinose were synthesized and then used as template analytes. The adsorption capacities of fructose, galactose and glucose-phenylboronic acid imprinted polymers were 75, 10 and 30%, respectively. The batch rebinding studies and Scatchard analysis were done for all sugar imprinted polymer. Glucose is one of the mostly found sugar in the urine. The glucose:phenylboronic acid imprinted polymer was used for the analysis of glucose, fructose, galactose, sucrose, maltose, lactose and raffinose in spiked urine. The selectivity of glucose:phenylboronic acid imprinted polymer to urine monosaccharides was found as nearly 45-55% and to di- and polysaccharides was found as 30-35%, respectively.

Immobilization of pancreatic lipase on chitin and chitosan.

Abstract In this study, porcine pancreatic lipase (EC 3.1.1.3) was immobilized on chitin and chitosan by adsorption and subsequent crosslinking with glutaraldehyde, which was added before (conjugation) or after (crosslinking) washing unbound proteins. Conjugation proved to be the better method for both supports. The properties of free and immobilized enzymes were also investigated and compared. The results showed that the pH optimum was shifted from 8.5 to 9.0 for both the immobilized enzymes. Also, the optimum temperature was shifted from 30 to 40°C for chitin‐enzyme and to 45°C for chitosan‐enzyme conjugates. The immobilization efficiency is low, but the immobilized enzymes have good reusability and stability (storage and operational). Besides these properties, the immobilized lipases were also suitable for catalyzing esterification reactions of fatty acids and fatty alcohols, both with a medium chain length. According to our results, esterification activities of immobilized lipases were two‐ and four‐fold higher for chitosan‐ and chitin‐enzyme, than for the free enzyme, respectively. The immobilization procedure shows a great potential for commercial applications of the immobilized lipase, a relatively low cost commercial enzyme.

ENCAPSULATION OF INSULIN IN CHITOSAN-COATED ALGINATE BEADS: ORAL THERAPEUTIC PEPTIDE DELIVERY

Insulin was encapsulated in calcium alginate beads coated with chitosan. Its release from alginate-chitosan and alginate-chitosan-glutaraldehyde beads was studied in artificial gastric (pH 1.2) and intestinal (pH 7.5) fluids. By comparing the release amounts, the ionic interaction between alginate-chitosan matrix with the medium pHs, intestinal fluid was found to be the better. The degradation of released insulin was also searched, even after 6 h incubation, the beads remained stable and the undegraded insulin seemed to be sufficient for the physiological conditions. Consequently, it can be said that the system can be offered for oral delivery of the therapeutic peptide drug insulin.Insulin was encapsulated in calcium alginate beads coated with chitosan. Its release from alginate-chitosan and alginate-chitosan-glutaraldehyde beads was studied in artificial gastric (pH 1.2) and intestinal (pH 7.5) fluids. By comparing the release amounts, the ionic interaction between alginate-chitosan matrix with the medium pHs, intestinal fluid was found to be the better. The degradation of released insulin was also searched, even after 6 h incubation, the beads remained stable and the undegraded insulin seemed to be sufficient for the physiological conditions. Consequently, it can be said that the system can be offered for oral delivery of the therapeutic peptide drug insulin.

Noncovalently galactose imprinted polymer for the recognition of different saccharides.

Molecularly imprinted polymers (MIPs) represent a new class of materials possessing high selectivity and affinity for the target molecule. The main goal of this study was to prepare a galactose imprinted polymer and its potential application for the recognition of different saccharides. The selectivity of galactose imprinted polymer for several saccharides; glucose, mannose, fructose, maltose, lactose, sucrose and raffinose was investigated. Macroporous polymer was prepared utilizing ethyleneglycoldimethacrylate as a crosslinking agent, in the presence of galactose as a template molecule with acrylamide as a functional monomer. After the synthesis of polymer, galactose was removed by methanol:acetic acid washing. The selectivity of galactose imprinted polymer for other saccharides was utilized by batch rebinding assay. The arrangement of functional groups within cavities versus shape selectivity is discussed. The results showed that, the orientation of the functional groups was the dominating factor for the selectivity of galactose imprinted polymer. The dissociation constants of polymer were determined by Scatchard analysis.

Preparation and Properties of α‐Galactosidase Chemically Attached to Activated Chitin

α‐Galactosidase (α‐D‐galactoside galactohydrolase, EC 3.2.1.22) from watermelon was covalently immobilized on chitin. The immobilized α‐galactosidase exhibited an activity of 0.61 U per g of carrier and an activity yield of 67%. The properties of free and immobilized α‐galactosidase were also searched and compared. The results showed that, optimum conditions for activity were not affected by immobilization. The optimum pH and temperature for free and immobilized enzyme found as pH 6.0 and 65°C, respectively. Compared with the free enzyme, the temperature and pH stabilities of the immobilized enzyme were similar. Both the enzymes were stable between pH 2–10 and below 50°C. The Km values for free and immobilized enzyme were determined using p‐nitrophenyl‐α‐D‐galactopyranoside (PNPG) and raffinose as substrates. Operational stability of the immobilized enzyme was investigated by using both substrates. The operational half‐life (t 1/2) was calculated as 34 h for PNPG and 28 h for raffinose. The immobilized α‐galactosidase was also utilized in the hydrolysis of raffinose. The immobilization procedure on chitin was cheap and also easy to carry out, and the immobilized enzyme had good properties that the potential for practical application is considerable.

Immobilization and stabilization of α-galactosidase on Sepabeads EC-EA and EC-HA

α-Galactosidase from tomato has been immobilized on Sepabead EC-EA and Sepabead EC-HA, which were activated with ethylendiamino and hexamethylenediamino groups, respectively. Two strategy was used for the covalent immobilization of α-galactosidase on the aminated Sepabeads: covalent immobilization of enzyme on glutaraldehyde activated support and cross-linking of the adsorbed enzymes on to the support with glutaraldehyde. By using these two methods, all the immobilized enzymes retained very high activity and the stability of the enzyme was also improved. The obtained results showed that, the most stable immobilized α-galactosidase was obtained with the second strategy. The immobilized enzymes were characterized with respect to free counterpart. Some parameters effecting to the enzyme activity and stability were also analyzed. The optimum temperature and pH were found as 60°C and pH 5.5 for all immobilized enzymes, respectively. All the immobilized α-galactosidases were more thermostable than the free enzyme at 50°C. The stabilities of the Sepabead EC-EA and EC-HA adsorbed enzymes treated with glutaraldehyde compared to the stability of the free enzyme were a factor of 6 for Sepabead EC-EA and 5.3 for Sepabead EC-HA. Both the free and immobilized enzymes were very stable between pH 3.0 and 6.0 and more than 85% of the initial activities were recovered. Under the identical storage conditions the free enzyme lost its initial activity more quickly than the immobilized enzymes at the same period of time. The immobilized α-galactosidase seems to fulfill the requirements for different industrial applications.

Comparison of Chitin and Amberlite IRA‐938 for α‐Galactosidase Immobilization

Watermelon α‐galactosidase (EC 3.2.1.22) was immobilized on a natural (chitin) and a synthetic anion‐exchange (Amberlite IRA‐938) support by covalent coupling methods. The procedure entails the activation of supports with 1,1′‐carbonyldiimidazole (CDI), followed by immobilization of the enzyme on to these supports without and with a spacer arm; γ‐aminobutyric acid (GABA). Optimization of activation was performed by changing the CDI concentrations and coupling efficiencies. The comparison of two immobilization techniques for both chitin and Amberlite IRA‐938 was made by comparing different enzyme concentrations against enzyme activity yield. Furthermore, the storage stability of the immobilized enzymes was also investigated and chitin immobilized α‐galactosidase was found to be better. Although the activity yield of immobilized enzymes were the same for both supports, the short storage stability of immobilized enzyme on Amberlite IRA‐938 is currently a drawback to its applications.

Immobilization of Avocado Phytase on Epoxy-Activated Sepabead EC-EP and its Application in Soymilk Phytate Hydrolysis

There is a great demand for using phytases to reduce phytate content in animal feed stuffs and food for human consumption. Industrial application of phytase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to the free enzyme. In the present study, phytase was immobilized on Sepabead EC-EP and then used in the biodegradation of soymilk phytate. The immobilized enzyme exhibited an activity of 0.1 U per g of carrier and activity yield of 70.83%. Optimum temperature and pH for the immobilized enzyme were 55°C and 5.5, respectively. Both the enzymes were stable between pH 3.0–8.0 and below 70°C. Kinetic parameters(Km and Vmax) and the usability of the immobilized enzyme were determined. The immobilized enzyme hydrolyzed 65% of soymilk phytate in 8 h at 60°C, as compared with 56% hydrolysis observed for the native enzyme over the same period of time.

Immobilization of Phospholipase A2 on Porous Glass and Its Application for Lowering Serum Cholesterol Concentration

Phospholipase A2 (PLA2; EC 3.1.1.4) is a lipolytic enzyme that hydrolysis the ester bond in sn‐2 position of phospholipids. In this work, the PLA2 from hog pancreas was covalently coupled to porous glass. The properties of free and immobilized enzyme were also investigated and compared. The optimum pH and temperature were found as 8.5 and 50°C, respectively for both free and immobilized enzyme. The immobilized enzyme had good properties that potential for medical application is considerable. Its use in lowering plasma cholesterol concentrations in blood samples was also demonstrated.