Enzymatic reduction of cystine

Abstract

The present invention relates to a method for the enzymatic reduction of cystine to cysteine comprising contacting cystine with a reduction solution comprising: (i) an active glutathione reductase (EC1.8.1.7); (ii) a cofactor; and (iii) glutathione; and recovering a cysteine comprising composition, wherein the reduction solution has a pH of at least 6 during contacting with cystine.

Claims

1. A method for enzymatic reduction of cystine to cysteine comprising contacting cystine with a reduction solution comprising: (i) purified active glutathione reductase (EC1.8.1.7); (ii) a cofactor; and (iii) purified glutathione; and recovering a cysteine comprising composition, wherein the reduction solution has a pH of at least 6 during contacting with cystine.

2. The method according to claim 1, wherein said recovering the cysteine comprising composition is carried out within 15 hours after contacting cystine with the reduction solution.

3. The method according to claim 1, wherein the cofactor is nicotinamide adenine dinucleotide phosphate (NADPH), optionally in oxidized form (NADP+) or nicotinamide adenine dinucleotide (NADH), optionally in oxidized form (NAD+).

4. The method according to claim 1, wherein the reduction solution further comprises: (iv) a cofactor regeneration system comprising glucose dehydrogenase and glucose or formate dehydrogenase and formate.

5. The method according to claim 1, wherein the molar ratio of the glutathione: cystine in the reduction solution is less than 0.1.

6. The method according to claim 1, wherein the reduction solution has a pH within the range of 7.0 to 8.5.

7. The method according to claim 1, wherein the cystine is contacted with the reduction solution for a time period which is sufficient to reduce more than 50% of the cystine to cysteine.

8. The method according to claim 1, wherein contacting the cystine with the reduction solution is carried out under anaerobic conditions.

9. The method according to claim 1, wherein the cysteine comprising composition comprises cysteine and one or more selected from gluconic acid, formic acid, glucose, formate, nicotinamide adenine dinucleotide phosphate, nicotinamide adenine dinucleotide, glutathione and glutathione reductase.

Description

FIGURE LEGENDS

(1) FIG. 1: Percentage reduced L-Cystine in several reaction setups with varying glutathione concentrations and NADH as cofactor.

(2) FIG. 2: Percentage reduced L-Cystine in several reaction setups with varying glutathione concentrations and NADPH as cofactor.

(3) FIG. 3: pH activity curves for yeast Glutathione reductase. Activities are corrected for the background conversion of NADPH at the different acidities and expressed relative to the highest activity observed between pH 5 and 10.

(4) FIG. 4: formation of cysteine in time using yeast GR under different process conditions

(5) FIG. 5: Glutathione reductase activity before and after incubation at 55° C.

MATERIALS AND METHODS

(6) 1. Materials

(7) The following materials where used in the examples.

(8) L-Cystine, Sigma-aldrich, C7602-25G

(9) Glutathione Reductase from baker's yeast (S. cerevisiae), Sigma-aldrich, G3664-2.5KU

(10) L-Glutathione reduced, Sigma-aldrich, G4251-100G

(11) L-Glutathione oxidized, Sigma-aldrich, G4376-10G

(12) Thioredoxin Reductase from Escherichia coli, Sigma-aldrich, T7915-250UG

(13) Thioredoxin from Escherichia coli, Sigma-aldrich, T-0910-1 MG

(14) B-Nicotinamide adenine dinucleotide phosphate, reduced tetra (cyclohexylammonium) salt, Sigma-aldrich, N5130-25MG

(15) B-Nicotinamide adenine dinucleotide reduced disodium salt hydrate, Sigma-aldrich, N8129-50MG

(16) B-Nicotinamide adenine dinucleotide phosphate hydrate, Sigma-aldrich, N5755-110MG

(17) B-Nicotinamide adenine dinucleotide hydrate, Sigma-aldrich, N1636-100MG

(18) Glucose dehydrogenase from Pseudomonas sp., Sigma-aldrich, 19359-10MG-F

(19) Formate dehydrogenase from Candida boidinii, Sigma-aldrich, F8649-50UN

(20) D(+)-Glucose, Anhydrous, Merck, CAS 50-99-7, Calbiochem

(21) Sodium formate, Sigma-aldrich, 71539-500G

(22) di-sodium hydrogen phosphate dihydrate, Merck, CAS No 10028-24-7

(23) sodium dihydrogen phosphate monohydrate, Merck, CAS No 10049-21-5

(24) 2. Reaction

(25) The reactions of the present examples are carried out in a reduction solution which is a sodium phosphate buffer. First a saturated L-cystine solution was made by stirring >500 mg/L L-cystine in sodium phosphate buffer (pH 6.0 or pH 8.0) for 1 hour at room temperature. Further the additional optional ingredients such as thiol reducing enzyme, mediator, cofactor and cofactor regeneration system were added. The reaction was carried out at room temperature (20-25° C.) for 2 hours. The reaction was stopped by adding a deuterium oxide solution comprising 40 g/l malic acid, 100 mg/I 1.1-difluoro-1-trimethylsilanyl methylphosphonic acid (FSP), having a pH of 6.4 with 50% NaOH and incubation for 1 hour.

(26) 3. NMR Analysis

(27) The obtained reduction solution comprising L-cysteine was analysed by NMR for L-cystine and free thiol groups. Samples were measured in 3 mm NMR tubes on a 700 MHz spectrometer equipped with a helium-cooled cryoprobe. NOESYGPPR1d.COMP water suppression was applied. 32 Scans were acquired with a relaxation delay of 1.2 seconds. Components were quantified according to the integrals of the peaks relative to the peak area of FSP at 0 ppm:

(28) D 5.04 ppm: glucose

(29) Dd 3.18 ppm: Cystine

(30) Dd 3.09 ppm: GSSG

(31) M 2.24 ppm: total SH.

(32) 4. % Cystine Reduction

(33) The percentage (%) reduction of cystine was calculated by subtracting the amount of cystine at the end of the reaction from the amount of cystine at the start of the reaction, and dividing this number by the amount of cystine at the start of the reaction, multiplied with 100.

EXAMPLES

Example 1

(34) Ratio's Mediator: L-Cystine

(35) 175 μl L-cystine solution (pH 8.0) was pipetted to a 96-well plate. Five μl of the following were added to a final reaction volume of 200 μl: glutathione reductase (17 U/ml); NADH or NADPH (1.0 and 0.28 mM); glucose dehydrogenase (3.4 U/ml) and D-glucose (2 mM); if applicable Milli-Q™ water. Finally five μl of different glutathione concentrations were added (see FIGS. 1 and 2) to a final reaction volume of 200 μl. The final L-cystine concentration in the reaction was 0.85 mM final reaction. Reactions were executed as described in the materials and methods and the L-cystine and free thiol groups were analyzed by NMR.

(36) FIG. 1 shows the percentage of reduced L-cysteine with varying glutathione concentrations. Specifically, FIG. 1 shows that the non-enzymatic reduction in the first series with only GSH and NADH provides a reduction above 90% only if much more GSH than L-cystine is present. Surprisingly, after addition of glutathione reductase, an increased reduction is obtained at lower ratio's of GSH:L-cystine.

(37) Further, FIG. 1 shows that with the presence of a cofactor regeneration system like glucose dehydrogenase and D-glucose even lower amounts of mediator are needed to provide a reduction above 90%, surprisingly enabling a significant reduction of the ratio of GSH:L-cystine. Moreover, the added cofactor regeneration system is able to reduce cystine at a reduction above 90% at much lower cofactor NADH concentrations (since 0.28 mM NADH results in the same reduction towards L-cysteine as with an excess amount of 1.0 mM NADH) as compared to without a cofactor regeneration system.

(38) Analogous to FIG. 1, FIG. 2 discloses that a reduction of L-cystine towards L-cysteine of higher than 90% can be obtained with NADPH as cofactor, even if the ratio of glutathione: L-cystine is less than 1. Here, the addition of a cofactor regeneration system is able to reduce cystine at a reduction above 90% not only at much lower cofactor NADPH concentrations (since 0.28 mM NADPH results in the same reduction towards L-cysteine as with an excess amount of 1.0 mM NADPH) as compared to without a cofactor regeneration system, it also surprisingly allows for an even further reduction of the ratio of GSH:L-cystine.

(39) To conclude, FIG. 1 and FIG. 2 disclose the feasibility of the present invention in providing an improved method for the reduction of L-Cystine.

Example 2

(40) Reduction Using Cell Free Extract (Abbreviated as CFE)

(41) CFE Preparation

(42) Yeast (Saccharomyces cerevisiae) and fungal (Penicillium chrysogenum) cell cultures were washed with Milli-Q™ water, suspended in 100 mM Tris-HCL buffer (pH 7.5) and stored on ice. Cells were transferred to 2.0 ml vials containing 0.5 g of 0.45-0.5 mm glass beads and thoroughly shaken three-times at 5000 rpm for 40 seconds on a Precellys homogenizer with cooling on ice in between. Extract was centrifuged twice, while transferring supernatant to fresh tubes after each centrifugation step. Total protein content of cell free extract (CFE) was determined according to the Biuret Method. Samples were stored at −20° C. until further use.

(43) Assay:

(44) 185 μl L-Cystine solution (pH 6.0 or 8.0) was pipetted to a 96-well plate. Five μl of the following were added to a final reaction volume of 200 μl: yeast (1 mg/ml) or fungal (0.5 mg/ml) CFE; mediator L-glutathione (100 μg/ml); NADH (700 μg/ml) or NADPH (800 μg/ml); if applicable Milli-Q™ water. L-cystine concentrations were 0.63 mM at pH6.0 and 0.78 mM at pH 8.0 in the final reaction. Reactions (see Table 2) were executed as described in the materials and methods and the L-cystine and free thiol groups were analyzed by NMR.

(45) TABLE-US-00001 TABLE 2 Overview of L-Cystine reduction reactions using CFE and percentage reduction Substrate pH CFE mediator co-factor Reduction L-cystine 6 S. cerevisiae — NADH 0 GSH 32 — NADPH 1 GSH 33 Penicillium chr. — NADH 0 GSH 30 — NADPH 0 GSH 37 8 S. cerevisiae — NADH 6 GSH 31 — NADPH 0 GSH 100 Penicillium chr. — NADH 7 GSH 47 — NADPH 2 GSH 72

(46) Table 2 clearly shows that at pH 8 the reduction of L-cystine is increased. Further, it is clear from Table 2 that CFE's of S. cerevisiae and Penicillium chr. are able to provide the reduction of L-cystine.

Example 3

(47) Co-Factor Regeneration

(48) 175 μl L-cystine (pH 8.0) was pipetted to a 96-well plate. Five μl of the following were added to a final reaction volume of 200 μl: glutathione reductase (17 U/ml) or yeast CFE (1 mg/ml); mediator L-glutathione (100 μg/ml); NADH or NADPH (200 and 350 μg/ml); glucose dehydrogenase (GDH) or formate dehydrogenase (FDH) (both 3.4 U/ml); D-glucose or sodium formate (both 1 mM); if applicable Milli-Q™ water. L-cystine concentrations were 0.80 mM and 0.52 mM in purified enzyme and CFE reactions respectively. Reactions (see Table 3) were executed as described in the materials and methods and the L-cystine and free thiol groups were analyzed by NMR.

(49) TABLE-US-00002 TABLE 3 Reaction setup for co-factor regeneration with purified enzyme or CFE and percentage of reduced L-Cystine. Co- Reducing Regenerating Regenerating Substrate Mediator factor enzyme enzyme substrate Reduction % L-cystine GSH NADH — GDH glucose 10 glutathione — — 14 reductase GDH glucose 91 FDH formate 100 NADPH — GDH glucose 15 glutathione — — 30 reductase GDH glucose 100 FDH formate 100 NADH Yeast — — 9 CFE glucose 0 formate 6 GDH glucose 28 FDH formate 100 NADPH — — 55 glucose 68 formate 68 GDH glucose 100 FDH formate 79

(50) Table 3 clearly shows that by using a cofactor regeneration system the reduction of cystine is obtained, while only limited amounts of cofactor NADH or NADPH are used (200 and 350 μg/ml, whereas in example 1 and 3 700 and 800 μg/ml are used, respectively). Further, Table 3 shows that by using cell free extract in combination with cofactor regeneration 100% reduction of cystine is obtained.

Example 4

(51) Oxidized Cofactor

(52) 175 μl L-cystine solution (pH 8.0) was pipetted to a 96-well plate. Five μl of the following were added to a final reaction volume of 200 μl: glutathione reductase (17 U/ml); mediator L-glutathione (1 mM); NADH, NAD+, NADPH or NADP+ (all 1 mM); glucose dehydrogenase (3.4 U/ml) and D-glucose (2 mM); if applicable Milli-Q™ water. L-cystine concentration was 0.96 mM in final reactions. Reactions (see Table 4) were executed as described in the materials and methods and the L-cystine and free thiol groups were analyzed by NMR.

(53) TABLE-US-00003 TABLE 4 Reactions with reduced-and oxidized co-factor and percentage of reduced L-Cystine. Reducing Co- Regenerating Regenerating Reduction Substrate enzyme Mediator factor enzyme substrate (%) L-Cystine Glutathione GSH NADH Glucose D-Glucose 100 Reductase NAD+ Dehydrogenase 100 NADP+ 100 NADPH 100

(54) Table 4 shows that using a cofactor in oxidized form results in a 100% reduction of cystine.

Example 5

(55) pH Dependent Activity of Yeast Glutathione Reductase

(56) The pH curve for yeast Glutathione Reductase (GR) enzyme was obtained by assaying the activity at every pH unit between pH 5 and 10 in 0.1 M phosphate buffer. A blank without GR enzyme was included at every pH. The buffers for pH 5-9 were mix buffers of 0.1 M Na2HPO4 solution and 0.1M NaH2PO4 solution. Addition of sodium hydroxide was required for reaching a buffer pH 10. An NADPH stock of 50 mM was prepared in milliQ water with added sodium hydroxide to pH 8, where NADPH is soluble and stable. The GSSG solutions were prepared separately for each pH (20 mM=61.3 mg/5 mL buffer in 15 mL Greiner tube). The enzyme was first diluted 20× in water and then 100× in buffer at the different pH set points. The NADPH stock solution was diluted 25× in the desired buffer briefly before starting the assay to minimize the background conversion of NADPH before addition of enzyme. The assay was performed as follows: 100 microliter GSSG solution pH X+50 microliter NADPH solution pH X+50 microliter diluted enzyme pH X. Subsequently, a kinetic readout with decrease in absorption at 340 nm per minute at desired pH was carried out. The slopes of the reactions (Δ340 nm/min) were corrected for the slope of the blank reaction at the same pH and activities were expressed relative to the highest measured activity for the enzyme. The relative activity is shown in FIG. 3.

Example 6

(57) Cystine Reduction in Presence of Enzyme at Different Process Conditions.

(58) Reduction of cystine to cysteine was carried out in presence of oxidized glutathione as mediator and NADP+ as cofactor using yeast glutathione reductase (sigma) and Glucose dehydrogenase in order to regenerate NAPPH in the reaction. The schematic reaction is as follows:
CYS−CYS+2GSH.fwdarw.2CYS+GSSG  Reaction 1:
GSSG+NADPH.fwdarw.2GSH+NADP+H  Reaction 2:
Glucose+NADP.fwdarw.Gluconic acid+NADPH  Reaction 3:

(59) The enzymatic conversion was performed in a 200 ml jacketed vessel with temperature and pH control. The total working volume was 100 ml. Reactions were performed in two vessels. Both vessels contained yeast GR (Sigma) and Glucose dehydrogenase (GDH 105, Codexis). Reactions were performed two different process conditioned indicated in Table 5 below.

(60) TABLE-US-00004 TABLE 5 Amounts of components and process conditions applied in reduction of Cystine using yeast GR and GDH enzymes (in 100 mL) compounds Vessel 1 Vessel 2 Yeast GR (Sigma) 2 units/ml 2 units/ml GDH 105 (Codexis) 2 units/ml 2 units/ml GSH (g) 0.12 0.12 Cystine (g) 1.0 1.0 NADPH (g) 0.0076 0.0076 Temperature (° C.) 30 51 pH 8.0 5.1

(61) Samples were taken at t=0 (before addition of enzyme), 0.5, 1, 2, 3, 4, 5, and 6 hours. 200 μl sample was centrifuged 1 minute at 13.000 RPM and 100 μl supernatant was transferred to 900 μl 0.111N HCL (final concentration of 0.1N HCL), mixed and stored at −20° C. All samples were analyzed by LCMS-MS Method to measure L-cystine, L-cysteine, GSH, GSSG and combinations of these components. The cysteine formation in time is illustrated in FIG. 4.

(62) The Level of cysteine in vessel 2, did not increase in time showing that the yeast glutathione reductase was not active under applied conditions of 51° C. and pH 5.1. The initial increase of cysteine in this reaction is the result of non enzymatic reduction with GSH and does not further increase as glutathione reductase is not active and thus cannot reduce the oxidized glutathione.

Example 7

(63) Stability of Glutathione Reductase at 55° C. Temperature

(64) To study the heat stability of glutathione reductase, 1.5 ml of enzyme stock solution was incubated at 55° C. After 15, 30, 60, 120 and 240 minutes of incubation, 250 μl was transferred to a fresh tube and stored in ice-water. The non-incubated stock solutions (˜3.5 ml) were directly placed on ice-water, without additional incubation.

(65) The following reaction was performed:
NADPH+GR+GSSG.fwdarw.NADP.sup.++GR+2GSH

(66) In the reaction mixture 50 μl Tris-HCL buffer (pH 8.0), 50 μl NADPH, 50 μl glutathione reductase (GR), and 50 μl glutathione oxidized were added. As glutathione reductase, either a non-incubated or heat-treated sample were used. Reactions were carried out in total volume of 200 μl in a flat, clear bottom MTP96 plate. The reactions were carried out at room temperature. All components were mixed except for glutathione. To start the reactions simultaneously, these substrates were added just before placing the MTP96 plate in the μquant spectrophotometer. Absorbance was measured every minute for two hours, at 340 nm.

(67) FIG. 5 shows the results of absorbance in time after incubation at higher temperature. The activity already declines after 15 minutes and no activity is measured after 30 and 60 minutes incubation at 55° C.

Example 8

Yeast Glutathione Reductase Thermostability in Presence and Absence of Protease

(68) 20 Units of Yeast Glutathione reductase (Sigma) was added in 10 ml buffer (0.5M Tris HCL buffer) and incubated at 51° C. and pH 5.1 for 18 hours with and without protease in a water bath. Protease applied in this experiment was Alcalase (Novozyme) at the dosage of 0.0068 mg in gram dry mater of the solution. The Enzyme activity was measured after incubation in both solutions using developed assay by spectrophotometer.

(69) TABLE-US-00005 TABLE 6 GR activity in the buffer solution before and after incubation with and without Alcalase Sample Before (t = 0) After (t = 18 h) Yeast GR without Alcalase 2 U/ml 0.010 U/ml Yeast GR with Alcalase 2 U/ml 0.015

(70) Results showed that yeast GR enzyme is not thermostable and the activity is lost after incubation at 51 degrees Celsius and pH 5.1 after 18 hours.

(71) Assay for Glutathione Reductase Activity Measurement

(72) The assay for measuring GR activity was based on the following reaction:
GR+GSSG+NADPH.fwdarw.GR+2GSH+NADP+

(73) Where GSSG=L-glutathione oxidized and GSH=L-glutathione reduced.

(74) Materials Applied were:

(75) Glutathione reductase assay solution:

(76) 20 mM NADPH (CAS number: 2646-71-1) in mili-Q (mQ)

(77) 20 mM GSSG (CAS number: 27025-41-8) in mQ

(78) 200 mM Tris-HCl (pH 7.5, obtained in DSM MBK)

(79) For preparing 45 ml stock solution (50 samples): 17 ml mQ, 22.5 ml 200 mM Tris-HCl (pH 7.5), 500 μl 20 mM NADPH, 5 ml 20 mM GSSG were mixed right before the assay.

(80) Method:

(81) The spectrophotometer was calibrated (types: Ultrospec® 2000 in Genetics and Jasco V-630 in ABC lab 1128) to 0.000 with air. the absorbance of assay stock solution was measured without sample. The result of the analysis was taken as negative control. the enzyme solution was Diluted in 100 mM Tris-HCl (pH 7.5). 100 μl of diluted sample was added to 900 μl assay stock solution in a cuvet and was mixed thoroughly with pipette. The cuvet was placed in the spectrophotometer and the absorbance of the solution was recorded at time zero (t=0) and after 1 minute (t=1).

(82) Calculations:

(83) The enzyme activity was determined by the formula below:

(84) $\mathrm{Activity}\left[U/\mathrm{ml}\right]=\frac{\mathrm{Df}*\Delta A{\min }^{-1}*\mathrm{Av}}{d*e*\mathrm{Sv}}$

(85) Where: Df=dilution factor ΔA=absorbance increase/decrease per minute min.sup.−1 Av=assay volume in ml (1 ml) d=optical path length in cm (for cuvet, this is 1 cm) e=molar extinction coefficient of NADPH, which is 6.22 M.sup.−1 cm.sup.−1 Sv=sample volume in ml (0.1 ml)