METHOD OF CONTINUOUSLY PRODUCING GLUTATHIONE USING PHOTOSYNTHETIC MEMBRANE VESICLES

Abstract

The present invention relates to a method of producing glutathione, wherein photosynthetic membrane vesicles and enzymes catalyzing glutathione synthesis are combined and glutamate, cysteine and glycine are used as reaction substrates. As enzymes catalyzing glutathione synthesis, γ-glutamylcysteine synthetase and glutathione synthetase may be used together, or bifunctional glutathione synthetase may be used alone. According to the conventional methods, there is a problem in that expensive adenosine triphosphate should be continuously supplied when glutathione is produced. However, according to the present invention, since photosynthetic membrane vesicles are used as a source to regenerate adenosine triphosphate, it is possible to continuously produce glutathione without additionally adding adenosine triphosphate, thereby reducing production costs of glutathione.

Claims

1. A method of producing glutathione, comprising: a) a step of generating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and an inorganic phosphate by irradiating photosynthetic membrane vesicles with light; and b) a step of synthesizing glutathione by enzymes that catalyze glutathione synthesis using ATP generated in step a), and forming ADP and an inorganic phosphate.

2. The method according to claim 1, further comprising c) a step of reusing ADP and an inorganic phosphate forming in step b) to generate ATP in photosynthetic membrane vesicles.

3. The method according to claim 1, wherein the photosynthetic membrane vesicles are selected from the group consisting of chromatophore membrane vesicles isolated from purple non-sulfur bacteria and thylakoid membrane vesicles isolated from cyanobacteria or algae.

4. The method according to claim 1, wherein the enzymes that catalyze glutathione synthesis are one or more selected from the group consisting of γ-glutamylcysteine synthetase (GSH-I), glutathione synthetase (GSH-II) and bifunctional glutathione synthetase (bifunctional γ-glutamylcysteine synthetase/glutathione synthetase, GshF).

5. The method according to claim 1, further comprising a step of adding glutamate, cysteine and glycine as substrates for producing glutathione.

6. The method according to claim 1, wherein step b) comprises a step of synthesizing γ-glutamylcysteine from glutamate and cysteine by γ-glutamylcysteine synthetase while converting adenosine triphosphate generated in step a) into adenosine diphosphate and an inorganic phosphate; and a step of synthesizing glutathione from the synthesized γ-glutamylcysteine and glycine by glutathione synthetase while converting adenosine triphosphate generated in step a) into adenosine diphosphate and an inorganic phosphate.

7. The method according to claim 1, wherein step b) comprises a step of synthesizing glutathione from glutamate, cysteine and glycine by bifunctional glutathione synthetase while converting adenosine triphosphate generated in step a) into adenosine diphosphate and an inorganic phosphate.

8. The method according to claim 6, wherein a relative activity ratio of γ-glutamylcysteine synthetase to glutathione synthetase is 4:1 to 20:1.

9. The method according to claim 6, wherein a relative activity ratio of the photosynthetic membrane vesicles to γ-glutamylcysteine synthetase to glutathione synthetase is 1:12:1 to 50:12:1.

10. The method according to claim 7, wherein a relative activity ratio of photosynthetic membrane vesicles to bifunctional glutathione synthetase is 10:1 to 500:1.

11. A composition for producing glutathione, comprising: i) photosynthetic membrane vesicles; and ii) enzymes that catalyze glutathione synthesis, wherein the enzymes are selected from the group consisting of γ-glutamylcysteine synthetase (GSH-I), glutathione synthetase (GSH-II) and bifunctional glutathione synthetase (bifunctional γ-glutamylcysteine synthetase/glutathione synthetase, GshF).

12. A system for continuously producing glutathione, comprising: i) photosynthetic membrane vesicles as a means for supplying adenosine triphosphate; ii) enzymes that catalyze glutathione synthesis, wherein the enzymes are selected from the group consisting of γ-glutamylcysteine synthetase (GSH-I), glutathione synthetase (GSH-II) and bifunctional glutathione synthetase (bifunctional γ-glutamylcysteine synthetase/glutathione synthetase, GshF), as a means for catalyzing glutathione synthesis

13. A method of continuously producing glutathione, the method comprising a step of adding glutamate, cysteine and glycine as substrates for producing glutathione to the system according to claim 12.

Description

DESCRIPTION OF DRAWINGS

[0045] FIG. 1a illustrates a schematic diagram (overall reaction A) showing a method of synthesizing glutathione using chromatophore membrane vesicles, γ-glutamylcysteine synthetase and glutathione synthetase. FIG. 1b illustrates a schematic diagram (overall reaction B) showing a method of synthesizing glutathione using chromatophore membrane vesicles and bifunctional glutathione synthetase.

[0046] FIG. 2 is an image showing the result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) performed on purified proteins of the present invention. Each band shown on the image represents purified γ-glutamylcysteine synthetase, glutathione synthetase or bifunctional glutathione synthetase.

[0047] FIG. 3 is a graph showing the activity of γ-glutamylcysteine synthetase measured using a method of detecting adenosine diphosphate formation.

[0048] FIG. 4 is a graph showing the activity of glutathione synthetase measured using a method of detecting adenosine diphosphate formation.

[0049] FIG. 5 is a graph showing the amount of glutathione produced depending on the relative activity of γ-glutamylcysteine synthetase while the relative activity of glutathione synthetase is fixed at 1.

[0050] FIG. 6 is a graph showing the amount of glutathione produced depending on the relative activity of chromatophore membrane vesicles while the relative activity of γ-glutamylcysteine synthetase to the relative activity of glutathione synthetase was fixed at 12:1.

[0051] FIG. 7 is a graph showing the activity of bifunctional glutathione synthetase for producing glutathione in a reaction solution containing L-glutamate, L-cysteine, glycine and adenosine triphosphate.

[0052] FIG. 8 is a graph showing the amount of glutathione produced depending on the relative activity of chromatophore membrane vesicles while the relative activity of bifunctional glutathione synthetase is fixed at 1.

MODES OF THE INVENTION

[0053] Hereinafter, the present invention is described in more detail with reference to the following examples. These examples are only intended to explain the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

EXAMPLES

Example 1: Isolation of Chromatophore Membrane Vesicles

[0054] Chromatophore membrane vesicles are isolated using the method described in Korean Patent Application No. 10-2014-0151907. Rhodobacter sphearoides (Rhodobacter sphearoides 2.4.1, ATCC BAA-808, Cohen-Bazire et al. 1956. J. Cell. Comp. Physiol. 49: 25-68), a type of purple non-sulfur bacteria, was used to isolate chromatophore membrane vesicles. The strain was cultured in Sistrom's minimal medium (Sistrom. 1962. J. Gen. Microbiol. 28: 607-616, Table 1). The culture method is as follows. First, a test tube containing 5 ml of the medium was inoculated with the strain, and was subjected to shaking culture at 30° C. and 250 rpm. When culture absorbance at 660 nm was about 2.0, the medium was subcultured in an 18-ml screw cap test tube to an initial absorbance of 0.05, and then the screw cap test tube was filled with a fresh medium and sealed to block the exposure to oxygen. Culture was performed under photosynthesis conditions. Specifically, the culture was performed for 18 hours in an incubator, wherein culture conditions were set as follows: temperature is maintained at 30° C. and light is irradiated by an incandescent lamp at a luminous intensity of 15 Watts/m.sup.2. After culture, 8 ml of the strain cultured in the 18 ml screw cap test tube was added to a 260 ml transparent bottle, and the remaining volume of the transparent bottle was filled with a fresh medium and sealed. The culture medium was subjected to anaerobic culture, as described above, at a temperature of 30° C. and at a luminous intensity of 15 Watts/m.sup.2 for 18 hours. Thereafter, the process of isolating chromatophore membranes was carried out in an anaerobic chamber (anaerobic chamber, model 10, Coy laboratory product). The gas composition in the anaerobic chamber is 90% nitrogen, 5% carbon dioxide and 5% hydrogen. The Rhodobacter sphearoides strain cultured in the 260 ml transparent bottle was subjected to centrifugation at 7,000 g and 4° C. for 10 minutes, and then a supernatant was discarded and a cell pellet was obtained. The cell pellet was resuspended in 4 ml of a phosphate buffer (10 mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4, pH 7.6), and a protease inhibitor mixture (protease inhibitor cocktail, Roche) was added thereto according to the manufacturer's instructions. In subsequent procedure, all samples were kept on ice. Next, cell lysis was performed using a sonicator (model VCX130, Sonics & Materials) under the following conditions: ultrasonic irradiation for 2 minutes with 100% amplification and then cooling in ice water for 2 minutes, and repeating this process three times. When irradiating ultrasonic waves, the cell pellets were cooled with ice water to prevent overheating. The lysed cells were subjected to centrifugation at 6,000 g and 4° C. for 10 minutes to obtain a supernatant, and then the supernatant was subjected to centrifugation at 200,000 g and 4° C. for 1 hour using an ultracentrifuge (Optima XE-90, Beckman Coulter). After centrifugation, a supernatant was removed, and a pellet containing chromatophore membrane vesicles was dissolved in 1 ml of a phosphate buffer to perform sucrose-density gradient centrifugation. A sucrose-density gradient was formed in the order of 8 ml of a 60% (w/v, dissolved in a phosphate buffer) sucrose solution, 1 ml of a 40% sucrose solution and 1 ml of a 20% sucrose solution from the bottom layer of an ultracentrifuge tube with a volume of 13.5 ml. 1 ml of the pellet containing chromatophore membrane vesicles was placed on the top layer, and ultracentrifugation was performed at 200,000 g for 4 hours. A reddish brown layer containing chromatophore membrane vesicles was located between a layer of the 20% sucrose solution and a layer of the 40% sucrose solution. The reddish brown layer was separated, and then diluted by addition of the same volume of a phosphate buffer. In addition, kanamycin was added at a concentration of 100 μg/ml to prevent the growth of common contaminants, and a protease inhibitor mixture was added according to the manufacturer's instructions. The mixture was anaerobically sealed and stored at 4° C.

TABLE-US-00001 TABLE 1 Composition of Sistrom's minimal medium for culturing Rhodobacter sphearoides Additives Final Concentration KH.sub.2PO.sub.4 20 mM NaCl 8.5 mM (NH.sub.4).sub.2SO.sub.4 3.78 mM L-Glutamate 0.67 mM L-Aspartic acid 0.25 mM Succinic acid 34 mM Nitrilotriacetic acid 1.05 mM MgCl.sub.2•6H.sub.2O 1.2 mM CaCl.sub.2•2H.sub.2O 0.23 mM FeSO.sub.4•7H.sub.2O 7 μM (NH.sub.4).sub.6Mo.sub.7O.sub.24 0.16 μM EDTA 4.7 μM ZnSO.sub.4•7H.sub.2O 38 μM MnSO.sub.4•H.sub.2O 9.1 μM CuSO.sub.4•5H.sub.2O 1.6 μM Co(NO.sub.3).sub.2•6H.sub.2O 0.85 μM H.sub.3BO.sub.3 1.8 μM Nicotinic acid 8.1 μM Thiamine hydrochloride 1.5 μM Biotin 41 nM

Example 2: Preparation of Genes Encoding Enzymes Involved in Glutathione Synthesis

[0055] To clone genes encoding γ-glutamylcysteine synthetase and glutathione synthetase of overall reaction A (FIG. 1a), respectively, and a gene encoding bifunctional glutathione synthetase of overall reaction B (FIG. 1b), polymerase chain reaction (PCR) was performed. In the case of γ-glutamylcysteine synthetase, SEQ ID NO. 1 and SEQ ID NO. 2 were used as a forward primer and a reverse primer, respectively. In the case of glutathione synthetase, SEQ ID NO. 3 and SEQ ID NO. 4 were used as a forward primer and a reverse primer, respectively. In both cases, the chromosomal DNA of Escherichia coli (Escherichia coli str. K-12 substr. MG1655) was used as a PCR template. In the case of bifunctional glutathione synthetase, SEQ ID NO. 5 and SEQ ID NO. 6 were used as a forward primer and a reverse primer, respectively, and the chromosomal DNA of Streptococcus agalactiae (Streptococcus agalactiae str. 2603V/R, ATCC BAA-611) was used as a PCR template. The recognition sequences of a restriction enzyme, Bsa I, and additional sequences recommended by IBA Co. were inserted at both ends of the gene fragments amplified by PCR, and the resulting sequences were ligated to expression vectors, pASK-IBA7plus (in the case of γ-glutamylcysteine synthetase and bifunctional glutathione synthetase) and pASK-IBA3plus (in the case of glutathione synthetase), provided by IBA Co. As a result, gene constructs encoding γ-glutamylcysteine synthetase and bifunctional glutathione synthetase, respectively, in which a strep-tag was attached at the N-terminal, and a gene construct encoding glutathione synthetase, in which a strep-tag was attached to the C-terminal, were obtained.

TABLE-US-00002 TABLE 2 Primers for amplifying gene encoding γ- glutamylcysteine synthetase SEQ ID NO. Direction Sequences 1 gshA-F 5′-AAAAAAGGTCTCTGCGCTTGATCCCGGACG TATCACA-3′ 2 gshA-R 5′-AAAAAAGGTCTCTTATCATCAGGCGTGTTT TTCCAGCC-3′

TABLE-US-00003 TABLE 3 Primers for amplifying gene encoding glutathione synthetase SEQ ID NO. Direction Sequences 3 gshB-F 5′-AAAAAAGGTCTCTAATGATCAAGCTCGGC ATCGT-3′ 4 gshB-R 5′-AAAAAAGGTCTCTGCGCTCTGCTGCTGTA AACGTGCTT-3′

TABLE-US-00004 TABLE 4 Primers for amplifying gene encoding bifunctional glutathione synthetase SEQ ID NO. Direction Sequences 5 gshF-F 5′-AAAAAAGGTCTCAGCGCATGATTATCGAT CGACTGTTAC-3′ 6 gshF-R 5′-AAAAAAGGTCTCGTATCATTATAATTCTG GGAACAGTTTAG-3′

Example 3: Purification of Enzymes that Catalyze Glutathione Synthesis Reaction

[0056] Escherichia coli strains BL21 (DE3) were transformed with the expression vectors prepared in Example 2, and transformed strains overexpressing γ-glutamylcysteine synthetase, glutathione synthetase and bifunctional glutathione synthetase, respectively, were obtained. The same method was used for purifying these enzymes. First, 5 ml of LB (Luria-Bertani) medium containing 50 μg/ml ampicillin was added to a test tube, and the transformed strain was inoculated in the test tube, followed by shaking culture at 250 rpm and 37° C. for 12 hours. The cells, then, were inoculated into a 1 L flask filled with 500 ml LB medium containing 50 μg/ml ampicillin. At the time of inoculation, an initial absorbance at 600 nm was adjusted to 0.05, and then shaking culture was performed at 250 rpm and 37° C. until absorbance reached 0.4. Anhydrotetracycline was added to the culture at a concentration of 0.2 μg/ml and shaking culture was further continued at 250 rpm and 30° C. for about 12 hours. After culture, cells were centrifuged at 4,000 g for 10 minutes, and a cell pellet was obtained, followed by suspension in 10 ml of buffer W (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA). A protease inhibitor mixture (Roche) was added thereto in an amount recommended by the manufacturer, and the cell pellet was lysed using a sonicator (Branson sonifier 250). Sonication was performed on the suspended cell pellet for 5 minutes at the intensity of output 3, followed by cooling in ice water for 5 minutes. This process was repeated three times. After cell lysis, centrifugation was performed at 6,000 g for 10 minutes to separate a supernatant containing water-soluble enzymes, and the enzymes were purified using strep-tag affinity chromatography. The strep-tag affinity chromatography was performed according to the manufacturer (IBA)'s recommended method. The purified enzymes were verified by 10% SDS polyacrylamide gel electrophoresis (FIG. 2). The expected molecular weights were about 58 kDa for γ-glutamylcysteine synthetase (Watanabe et al. 1986. Nucleic Acids Res. 14: 4393), about 35 kDa for glutathione synthetase (Gushima et al. 1984. Nucleic Acids Res. 12: 9299), and about 85 kDa for bifunctional glutathione synthetase (Janowiak and Griffith. 2005. J. Biol. Chem. 280: 11829-11839).

Example 4: Measurement of Adenosine Triphosphate Production Activity of Chromatophore Membrane Vesicles Under Light Irradiation

[0057] The quantification and activity measurement of chromatophore membrane vesicles were carried out according to the method described in Korean Patent Application No. 10-2014-0151907. Chromatophore membrane vesicles were quantified using a bacteriochlorophyll a (bch a) concentration. When measuring adenosine triphosphate (ATP) production activity, chromatophore membrane vesicles were used at a concentration of 0.25-0.5 μg bch a/ml. An appropriate amount of chromatophore membrane vesicles was added to a buffer containing 10 mM sodium phosphate (Na.sub.2HPO.sub.4), 2 mM potassium phosphate (KH.sub.2PO.sub.4), 10 mM magnesium chloride (MgCl.sub.2), and 0.4 mM adenosine diphosphate, and the mixture was allowed to react under anaerobic conditions in which a temperature was maintained at 30° C. and light having a luminous intensity of 15 Watts/m.sup.2 was irradiated by incandescent lamp. The amount of adenosine triphosphate produced over time was measured by an adenosine triphosphate detection kit (Sigma-Aldrich). The activity of chromatophore membrane vesicles was expressed as the amount of adenosine triphosphate produced per unit time (nmole ATP/min).

Example 5: Measurement of Enzyme Activity of γ-Glutamylcysteine Synthetase and Glutathione Synthetase

[0058] To measure enzyme activity of γ-glutamylcysteine synthetase and glutathione synthetase purified in Example 3, a method of measuring adenosine diphosphate formation (Seelig and Meister. 1985. Methods in Enzymol. 113: 379-390) was used. A reaction buffer containing 0.1 mM Tris (Tris-C1, pH 7.6), 10 mM magnesium chloride (MgCl.sub.2), 0.8 mM adenosine triphosphate (ATP), 2 mM phosphoenolpyruvate, 0.2 mM reduced nicotinamide adenine dinucleotide (NADH), 14.3 Unit/ml pyruvate kinase, and 14.3 Unit/ml lactic dehydrogenase was used for activity measurement. When measuring the activity of γ-glutamylcysteine synthetase, 10 mM L-glutamate and 10 mM L-cysteine were added to the reaction buffer. When measuring the activity of glutathione synthetase, 10 mM glycine and 1 mM γ-glutamylcysteine were added to the reaction buffer. The reaction temperature was 30° C. When γ-glutamylcysteine synthetase and glutathione synthetase perform enzymatic reactions, adenosine diphosphate is produced, and at the same time, an amount of reduced nicotinamide adenine dinucleotide (NADH) equivalent to that of the adenosine diphosphate is converted into the oxidized form (NAD.sup.+). Since NADH absorbs light at 340 nm and NAD.sup.+ does not absorb light at 340 nm, the activity of the two enzymes can be determined by measuring the decrease in absorbance at 340 nm over time. FIG. 3 is a graph showing the result of measurement of γ-glutamylcysteine synthetase activity, and FIG. 4 is a graph showing the result of measurement of glutathione synthetase activity. When the enzyme activity was calculated, the change in the concentration of NADH was determined using a molar extinction coefficient (6,220 M.sup.−1cm.sup.−1) at 340 nm, and it was assumed that one equivalent of the product of each enzyme was produced when one equivalent of NADH was consumed. The calculated amount of product per unit time (nmole product/min) is referred to as enzyme activity.

Example 6: Confirmation of Glutathione Production by γ-Glutamylcysteine Synthetase and Glutathione Synthetase

[0059] In this example, it was confirmed that glutathione was produced from L-glutamate, L-cysteine, glycine and adenosine triphosphate by γ-glutamylcysteine synthetase and glutathione synthetase. A reaction buffer containing 10 mM sodium phosphate (Na.sub.2HPO.sub.4), 2 mM potassium phosphate (KH.sub.2PO.sub.4), 10 mM magnesium chloride (MgCl.sub.2), 0.8 mM adenosine triphosphate (ATP), 10 mM L-glutamate, 10 mM L-cysteine, and 10 mM glycine was used. To prevent oxidation of the resulting glutathione, the reaction was performed at 30° C. under anaerobic conditions. A glutathione detection kit (GSH-Glo™ Glutathione Assay, Promega) was used to detect glutathione. The activity of each enzyme was measured using the method described in Example 5, and relative activity of each enzyme was adjusted based on the measured activity. FIG. 5 is a graph showing the amount of glutathione produced depending on the relative activity of γ-glutamylcysteine synthetase. At this time, the relative activity of glutathione synthetase was fixed at 1, and the relative activity of γ-glutamylcysteine synthetase was varied based on the relative activity of glutathione synthetase. As a result, it was confirmed that glutathione was produced by the two enzymes, and the minimum activity ratio (i.e., GSH-I:GSH-II of FIG. 5) between the two enzymes with the highest glutathione production was about 12:1. Thus, in the following example, the ratio of γ-glutamylcysteine synthetase to glutathione synthetase was used at 12:1.

Example 7: Confirmation of Glutathione Production by Chromatophore Membrane Vesicles, γ-Glutamylcysteine Synthetase and Glutathione Synthetase Under Light Conditions

[0060] In this example, it was confirmed that glutathione was produced by chromatophore membrane vesicles, γ-glutamylcysteine synthetase, and glutathione synthetase in the conditions in which adenosine triphosphate (ATP) was not supplied but light was irradiated. A reaction buffer containing 10 mM sodium phosphate (Na.sub.2HPO.sub.4), 2 mM potassium phosphate (KH.sub.2PO.sub.4), 10 mM magnesium chloride (MgCl.sub.2), 0.4 mM adenosine diphosphate (ADP), 10 mM L-glutamate, 10 mM L-cysteine, and 10 mM glycine was used, and the reaction was performed at 30° C. under anaerobic conditions. An incandescent lamp with a luminous intensity of 15 Watts/m.sup.2 was used as a light source. FIG. 6 is a graph showing the amount of glutathione produced depending on the relative activity of chromatophore membrane vesicles. At this time, the relative activity of γ-glutamylcysteine synthetase to the relative activity of glutathione synthetase was fixed at 12:1. As a result, it was confirmed that the amount of glutathione produced (or the rate of glutathione produced) was proportional to the amount of chromatophore membrane vesicles. In addition, when the relative activity of chromatophore membrane vesicles was 25 or more, the amount of glutathione produced was close to maximum.

Example 8: Measurement of Activity of Bifunctional Glutathione Synthetase on Glutathione Production

[0061] In this example, it was confirmed that glutathione was produced by bifunctional glutathione synthetase in a reaction solution containing L-glutamate, L-cysteine, glycine, and adenosine triphosphate. The composition of the reaction solution was 10 mM sodium phosphate (Na.sub.2HPO.sub.4), 2 mM potassium phosphate (KH.sub.2PO.sub.4), 10 mM magnesium chloride (MgCl.sub.2), 100 mM L-glutamate, 10 mM L-cysteine, 25 mM glycine, and 0.8 mM adenosine triphosphate (ATP). The reaction was performed at 30° C. FIG. 7 is a graph showing the activity (nmole glutathione/min) of bifunctional glutathione synthetase for producing glutathione, wherein the activity was determined by measuring the amount of glutathione produced over time in each reaction solution containing a different amount of bifunctional glutathione synthetase. Since a lag phase was present at an early stage of the reaction catalyzed by bifunctional glutathione synthetase, the activity was calculated based on the reaction rate of the linear region.

Example 9: Confirmation of Glutathione Production by Chromatophore Membrane Vesicles and Bifunctional Glutathione Synthetase Under Light Conditions

[0062] In this example, it was confirmed that glutathione was produced from L-glutamate, L-cysteine, and glycine when chromatophore membrane vesicles and bifunctional glutathione synthetase were used together under conditions in which adenosine diphosphate was added instead of adenosine triphosphate and light was irradiated. 0.4 mM adenosine diphosphate was added in place of 0.8 mM adenosine triphosphate in the reaction solution of Example 8. The reaction was performed under anaerobic conditions in which temperature is maintained at 30° C. and light was irradiated by an incandescent lamp with a luminous intensity of 15 Watts/m.sup.2. FIG. 8 is a graph showing the amount of glutathione produced depending on the relative activity of chromatophore membrane vesicles while the relative activity of bifunctional glutathione synthetase was fixed at 1. It was confirmed that the amount of glutathione produced was proportional to the amount of chromatophore membrane vesicles.

[0063] The present invention has been described in detail with reference to preferred embodiments. It will be apparent to those skilled in the art that the preferred embodiments are only illustrative and that the scope of the present invention is not limited thereto. Accordingly, the actual scope of the present invention will be defined by the appended claims and equivalents thereof.