Post-graduate theses
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| Identifier |
uch.chemistry.msc//2003touloupakis |
| Title |
Απομόνωση, χαρακτηρισμός και ακινητοποίηση του ενζύμου Αλλιινάση σε στοιβαγμένα διπλά υδροξείδια |
| Creator |
Touloupakis, Eleftherios
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| Abstract |
Garlic (Aliium sativum) is important because of the culinary value of its flavour and odour. These characteristics are created by chemical transformation of a series of volatile sulphur compounds generated by cleavage of relatively stable, odourless, S-alk(en)yl cysteine sulphoxide flavour precursors by the enzymes alliinase and lachrymatory-factor synthase. Allicin, a cysteine sulfoxide, has been shown to possess a variety of important biological effects (anti-inflammatory, antimicrobial, antithrombotic, anticancer and antimutagenic). The garlic protein alliinase (alliin lyase) is a dimmer of two identical subunits of 51.1kDa each. The enzyme is a glycoprotein which contains about 6% of neutral sugars; it was demonstrated that alliinase binds to Con A and can form a complex with garlic lectins. The lectin-alliinase complex offers a high stability to the enzyme and its enzymatic activity. The formation of a strong lectin-alliinase complex makes the isolation of pure alliinase difficult. Alliinase catalyses the production of allicin (S-(+)-2-propenyl-L-cysteine sulfoxide) from alliin [(2-propenyl)-2-propenethiosulfinate]. Alliinase and allicin stability is considerably dependent from the storage conditions. In the crude extracts of garlic, allicin easily gives a variety of chemical compounds. Because of the instability of the enzyme and the main product, the direct clinical trials are not possible. An alternative strategy could be the use of stabilised alliinase in combination with alliin as a pill or capsule. The available pills that are found in the market do not have a significant output in alicin and this is due to the loss of activity of the enzyme during the pill preparation. Therefore, stabilisation of alliinase in biocompatible inorganic clays, as layered double hydroxides, would help in the development of clinical applications. Layered double hydroxides are a large group of compounds with the chemical formula [M(II)(1-X)M(III)X(OH)2]Y+(An-Y/n)mH2O (LDH-A), where M(II) are the cations of double-charged metals, M(III) are the cations of triple-charged metals, An- are interlayer anions. The structure of these compounds is formed by alternating charged brucite-like layers of the composition [M(II)(1-X)M(III)X(OH)2](2)Y+ and the layers containing A- anions and water molecules at the same time. The presence of two fragments of nanoscale thickness which differ sharply in composition, structure, and chemical properties makes it possible to employ these compounds as nanoreactors for chemical reactions of interlayer molecules. Layered double hydroxides have attracted interest thanks largely to their ability to exchange their anions for other negatively charged species. This property has lead to their proposed use as ion-exchange materials and hosts for biologically active molecules. The hosting of biologically active molecules inside layered double hydroxides is attractive because LDH can act as a ‘chemical flak-jacket’, protecting the host from degradation. Additionally, the hosting of a negatively charged species could provide improved ways for drugs and genetic material to be introduced into cells. If ingested, the biomolecule-LDH nano-hybrid can move across the mucous membrane of the intestine into the bloodstream. The neutral hybrid can then enter cells by moving across the negatively charged cell-membrane without the repulsive electrostatic interactions that would be experienced by the guest anion alone. Aim of the present work is the purification, characterization and subsequent immobilisation of the protein alliinase in the layers of magnesium-aluminium hydroxide - LDH. Purified alliinase was extracted from bulbs of garlic and was purified by using hydroxylapatite and concanavalin A affinity columns. The enzymatic assay demonstrated that the enzyme was very active. Antibodies anti-alliinase were prepared. In order to investigate the binding of the oligosaccharides to alliinase and their role on the properties of the enzyme, treatment of native and denatured alliinase with PNGase F was carried out. Alliinase was immobilized on LDH and analyzed by using various techniques. The immobilized enzyme demonstrated a significant stability.
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| Issue date |
2003-00-01 |
| Date available |
2007-11-05 |
| Collection
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School/Department--School of Sciences and Engineering--Department of Chemistry--Post-graduate theses
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Type of Work--Post-graduate theses
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| Permanent Link |
https://elocus.lib.uoc.gr//dlib/d/5/f/metadata-dlib-2003touloupakis.tkl
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| Views |
215 |
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