Ingo Krest

Cysteine Sulfoxides and Alliinase Activity of Some Allium Species

The flavor precursors of 17 species belonging to the Alliaceae family were analyzed by HPLC, and results were evaluated with respect to the classification of species into their genus, subgenus, and section. Identification and quantification of these precursors were carried out by synthetic and natural reference materials. In addition, nine of these species were investigated in terms of their alliinase activity. Alliinase (EC 4.4.1.4) catalyzes the conversion of odorless (+)-S-alk(en)yl-L-cysteine sulfoxides into volatile thiosulfinates. Cysteine sulfoxides as well as alliinase activity were found in all investigated samples, and (+)-S-methyl-L-cysteine sulfoxide was most abundant. (+)-S-Propyl-L-cysteine sulfoxide was detected in only a few, not closely related, species. Analysis of the crude protein extract of nine species gave evidence that alliinase activities of samples were similar in terms of pH and temperature optimum, K(M) value, and substrate specificity. For all investigated protein extracts, the highest specific alliinase activity was found for (+)-S-(2-propenyl)-L-cysteine sulfoxide (alliin). The substrate specificity of these enzymes was not related to relative abundance of the cysteine sulfoxides. However, SDS-PAGE yielded some significant differences among species in terms of their total protein compositions. Species belonging to different subgenera exhibited a specific protein pattern with molecular masses between 13 and 35 kDa.

Immobilization of alliinase on porous aluminum oxide

Membrane filters prepared from porous aluminum oxide (Anopore) were investigated for their potential use as a durable support for enzymes. Alliinase (EC 4.4.1.4) was chosen as a model enzyme for immobilization experiments. To allow for smooth fixation, the enzyme was immobilized indirectly by sugar-lectin binding. Monomolecular layers of the lectin concanavalin A and alliinase were applied by self-assembling processes. As an anchor for these layers, the sugar, mannan, was covalently coupled to the membrane surface. This procedure exhibits several advantages: (i) enzyme immobilization can be carried out under smooth conditions; (ii) immobilization needs little time; and (iii) protein layers may be renewed.

Development of a biosensor specific for cysteine sulfoxides

S-Alk(en)yl cysteine sulfoxides have been observed in several plants, mainly belonging to the onion family (Alliaceae), which are of high commercial interest (e.g. garlic, Allium sativum). The quality of most garlic containing herbal remedies produced from garlic powder is determined by their content of the cysteine sulfoxide alliin. Therefore, a comprehensive method for the documentation of alliin amounts present in the fresh plant material through to the final remedy is desirable. The newly developed biosensoric method described in this paper was designed in order to fulfil these demands. In contrast to conventional HPLC-methods, neither a pre-column derivatization nor a chromatographic separation are required allowing a high throughput of samples. This technique is based on immobilized alliinase (EC 4.4.1.4), which was combined with an ammonia-gas electrode. The enzyme was either placed in a small cartridge or was immobilized in direct contact of the electrode surface giving detection limits of 3.7 x 10(-7) and 5.9 x 10(-6) M. Founded on these experiments, a pH-sensitive electrolyte/insulator/semiconductor (EIS) layer structure made of Al/p-Si/SiO(2)/Si(3)N(4) was also combined with immobilized alliinase. Measurements could be performed in a range between 1 x 10(-5) and 1 x 10(-3) M alliin. All sensors were operated in the flow-through modus. A high specificity for alliin could be demonstrated for the electrode and a number of garlic samples were analyzed. Results gained with the new method showed a good correlation with those obtained with conventional HPLC-methods. In addition, onion and a variety of wild Allium species were analyzed in order to determine the amount of isoalliin or total cysteine sulfoxides present, respectively.

Immobilization of enzymes on PTFE surfaces

Membranes and powders prepared from PTFE (polytetrafluorethylene) were investigated for their potential use as multifunctional supports for enzymes. The obtained bioactive materials are valuable for the construction of biosensors and enzyme reactors. To allow covalent coupling of enzymes to PTFE, the surface of the material was treated with elementary sodium followed by oxidation with ozone or hydrogen peroxide.%Derivatization steps were optimized in order to achieve highest enzyme loading and short reaction times. Alliinase (EC 4.4.1.4) and L-lactic dehydrogenase (EC 1.1.1.27) were chosen as model enzymes and were either immobilized by covalent coupling or fixed indirectly by a sugar-lectin binding. For the latter method, the sugar mannan was bound to the membrane surface as an anchor for layers of the lectin concanavalin A and the alliinase. Highest alliinase loading was achieved at 0.2 microg x cm(-2). Immobilization of alliinase via the lectin concanavalin A and a bifunctional epoxide gave the best long-term stability.%L-Lactic dehydrogenase was most sufficiently immobilized by using benzoquinone as spacer. These procedures show several advantages: 1) enzymes can be immobilized under physiological conditions, 2) an enzyme-multilayer can be achieved, and 3) protein layers are renewable.

Derivatization of Fluorinated Polymers and their Potential Use for the Construction of Biosensors

Membranes and powders prepared from fluorinated polymers (e.g., Teflon™, PTFE) were investigated for their potential use as multifunctional supports for proteins, enzymes and sugars. To allow covalent coupling of biomolecules to PTFE, the surface of the material was treated with elementary sodium followed by oxidation with ozone or hydrogen peroxide. Derivatization steps were optimized in order to achieve highest loading and short reaction times. The enzymes alliinase (EC 4.4.1.4) and L-lactic dehydrogenase (EC 1.1.1.27), the lectin concanavalin A, and the sugar mannan were chosen as model substances. Highest alliinase loading was achieved at 0.08 µg×cm−2.