CHEMOENZYMATIC DEGRADATION OF EPOXY RESINS

Abstract

Provided is a method for degrading epoxy resins by enzymatic route, in particular by adding an epoxy resin in a solvent followed by treatment with a glutathione S-transferase. A method for recycling a composite material comprising epoxy resin as well as the use of glutathione 5-transferase and eventually para-hydroxybenzoate hydroxylase for degrading epoxy resins are also provided.

Claims

1. A method for degrading an epoxy resin comprising the following successive steps: a) adding in a solvent an epoxy resin R based on: at least one aromatic compound R1 bearing at least two epoxide groups per molecule and comprising at least one aromatic ring bearing at least one glycidyloxy group, and at least one curing agent R2, to obtain mixture MA, b) treating enzymatically the mixture MA obtained after step a), the treatment comprising a step of contacting the mixture MA with a glutathione S-transferase to obtain a mixture MB.

2. The method according to claim 1, further comprising a step c) performed after step b) or concomitantly with step b), step c) consisting in an enzymatic treatment which comprises a step of contacting mixture MA or mixture MB with a para-hydroxybenzoate hydroxylase to obtain a mixture MC.

3. The method according to claim 1, wherein enzymatic treatment consisting in step b) or step b) and step c) is followed by a successive step d) which consists in treating chemically mixture MB or mixture MC, the chemical treatment comprising a step of contacting mixture MB or mixture MC with an aqueous solution of a strong Bronsted base to obtain a mixture MD.

4. The method according to claim 1, wherein step a) comprises swelling of the epoxy resin R in the solvent.

5. The method according to claim 1, wherein in step a) the solvent is chosen among the group consisting in an aqueous solution of disodium phosphate and an aqueous solution of phosphoric acid and phenol.

6. The method according to claim 1, wherein in step b) the glutathione S-transferase is issued from a N. aromaticivorans strain.

7. The method according to claim 2, wherein in step c) the para-hydroxybenzoate hydroxylase is a mutated enzyme.

8. The method according to claim 3, wherein the aqueous solution of a strong Bronsted base in step d) is a sodium hydroxide aqueous solution.

9. The method according to claim 1, wherein the aromatic compound R1 corresponds to a diglycidyl ether of bisphenol.

10. The method according to claim 1, wherein the curing agent R2 is an amine or an imidazole derivative.

11. The method according to claim 3, wherein mixture MD obtained after step d) is then further submitted to steps b) to d) as defined above.

12. The method according to claim 3, wherein mixture MD obtained after step d) is further submitted to steps a) to d) as defined above.

13. (canceled)

14. (canceled)

15. A method for recycling a composite material CM comprising the following steps: a′) providing a composite material comprising an epoxy resin R based on: at least one aromatic compound R1 bearing at least two epoxide groups per molecule and comprising at least one aromatic ring bearing at least one glycidyloxy group, and at least one curing agent R2, b′) treating the composite material CM obtained after step a′), the treatment comprising a step of degrading the epoxy resin R with the method as defined in claim 1, in which case the epoxy resin R in step a) is the composite material CM, c′) obtaining after step b′) the composite material with a reduced content in epoxy resin.

16. The method of claim 5, wherein in step a) the solvent is an aqueous solution of disodium phosphate.

17. The method of claim 2, wherein in step c) the para-hydroxybenzoate hydroxylase is a mutated enzyme presenting at least two point mutations L199V or L200V and Y385F.

18. The method of claim 9, wherein the aromatic compound R1 corresponds to a bisphenol A diglycidyl ether.

Description

EXAMPLES

[0280] Epoxy resins tested in the examples are as follows:

[0281] Resin R1 is obtained by polymerization of Bisphenol A diglycidyl ether (also noted BADGE or DGEBA) in the presence of 4,4′-Methylenebis(2,6-diethylaniline) and has the following general structure:

##STR00003##

[0282] Resin R2 is obtained by polymerization of Bisphenol A diglycidyl ether (also noted BADGE or DGEBA) in the presence of an imidazole derivative (5-ethyl-2-methylimidazole) and has the following general structure:

##STR00004##

[0283] In the examples, GST is added as a broth of lysed GST-expressing bacteria. It is produced following the process:

[0284] 1. growing GST-expressing strain and centrifuging to obtain the cell pellets;

[0285] 2. mixing cell pellets with phosphate buffer (0.1M pH=7.0) in a proportion of 1 g (of pellets)/10 ml (of buffer);

[0286] 3. Lysing cells by sonication in the phosphate buffer to obtain a broth containing the GST.

[0287] In the examples, PHBH is added as a broth of lysed PHBH-expressing bacteria. It is produced following the process:

[0288] 1. growing PHBH-expressing strain and centrifuging to obtain the cell pellets;

[0289] 2. mixing cell pellets with phosphate buffer (0.1M pH=7.0) in a proportion of 1 g (of pellets)/10 ml (of buffer);

[0290] 3. Lysing cells by sonication in the phosphate buffer to obtain a broth containing the PHBH.

[0291] In the examples, ET004 is added as a broth of lysed ET004-expressing bacteria. It is produced following the process:

[0292] 1. growing ET004-expressing strain and centrifuging to obtain the cell pellets;

[0293] 2. mixing cell pellets with phosphate buffer (0.1M pH=7.0) in a proportion of 1 g (of pellets)/10 ml (of buffer);

[0294] 3. Lysing cells by sonication in the phosphate buffer to obtain a broth containing the ET004.

[0295] A—Step a) Adding an Epoxy Resin to a Solvent

[0296] This step aims at swelling and potentially decrosslinking the epoxy resin in order to improve the accessibility to the key bond sites for the enzymes used in the following steps.

[0297] First, a series of solvents have been tested (water, an aqueous solution of Na.sub.2HPO.sub.4, an aqueous solution of H.sub.3PO.sub.4, an aqueous solution of ammonia, acetic acid, DMSO, DMF, hexane and phenol).

[0298] The test consists in soaking an epoxy resin R1 or R2 with a solvent for 80 days at room temperature in resin/solvent proportions of 0.1 g/1 ml. These preliminary assays were conducted to evaluate the behavior of the materials once exposed to these solvents.

[0299] The aqueous solution of Na.sub.2HPO.sub.4 was selected not only for its capability of promoting modification of the resin, but also because it is safe to handle, it is cheap and it can be recycled.

[0300] The mixture obtained after step a) has been studied to evaluate what is happening during this step.

[0301] Step a) has thus been performed on resin R2 as follows:

[0302] 1.sup.st Step: Preparing a solution of 0.21 g Resin R2+20.56 g Na.sub.2HPO.sub.4*12H.sub.2O (M.W. 358.14 g/mol). Add 98.05 g H.sub.2O, stir at 90° C. for 1 h;

[0303] 2.sup.nd Step: Evaporating the solution from previous step at 80° C. under vacuum until the mixture weight goes from 119 g to 55 g;

[0304] 3.sup.rd Step: Diluting the previous concentrated solution with 300 g of deionized H.sub.2O at 90° C.;

[0305] 4.sup.th Step: Evaporating at 80° C. under vacuum, until the weight of the mixture is reduced to 44 g. Afterwards, add 456 g of water at 90° C.

[0306] 5.sup.th Step: Centrifuging the solution from previous step in order to obtain a yellowish supernatant and two kinds of solids: one yellowish and similar to the original resin, and the other grayish, brittle and opaque.

[0307] By HPLC and MS analysis of the resulting mixture, it was observed that first step a) led to degradation of the resin. Products resulting from phosphorylation of the resin and/or ether bond cleavage of the resin could notably be identified.

[0308] B—Step b) Treating Enzymatically by Contacting Mixture MA with a Glutathione 5-Transferase

[0309] After performing step a) on resin R1 and R2 and obtaining respectively MA1 and MA2, step b) was performed according to the method described below in order to evaluate the activity of the enzyme issued from a N. aromaticivorans strain and named “NaLigE”.

[0310] Ethyl vanillin was also tested along with MA1 and MA2 as a model.

[0311] Step a) Preparation of mixtures MA1 and MA2:

[0312] 1.sup.st Step: Prepare a solution of 1.98 g Resin+50.65 g deionized H.sub.2O; +90.00 g Na.sub.2HPO.sub.4.12H.sub.2O, +189 g deionized H.sub.2O, at 60° C. until complete solubilization of the Na.sub.2HPO.sub.4.12H.sub.2O salt;

[0313] 2.sup.nd Step: Evaporate the solution from the previous step at 80° C. under vacuum to remove water, then add 300 g of deionized H.sub.2O, then evaporate again until complete removal of water;

[0314] 3.sup.rd Step: Repeat the 2.sup.nd step;

[0315] 4.sup.th Step: Add deionized H.sub.2O at 90° C. to the dry solid obtained after step 3;

[0316] 5.sup.th Step: Add 9.96 g β-Cyclodextrin to the previous solution (total weight 142 g), and mix;

[0317] 6.sup.th Step: Stir the mixture at 90° C. for two days;

[0318] 7.sup.th Step: Dilute the mixture to obtain a volume of 200 ml;

[0319] The mixture obtained after performing step 1 to 7 on resin R1 is noted MA1.

[0320] The mixture obtained after performing step 1 to 7 on resin R2 is noted MA2.

TABLE-US-00001 TABLE A Volume 2.1 wt. % 2.48 Aqueous Glutathione- or reduced wt. % solution S- mass of glutathione Glycine of NaOH Transferase ID Substrate substrate (ml) (ml) 3M enzyme 1 Ethyl vanillin (0.4 0.398 ml 0.175 1.452  0.5 ml 3 ml wt. % in a 0.1M phosphate buffer system at pH 7) 2 MA2  0.5 g 0.175 0.725 1.225 ml 3 ml 3 MA1  0.5 g 0.175 0.725 1.225 ml 3 ml

[0321] According to table A, in a reactor of suitable volume, the components were added in the following order: 1.sup.st: substrate (ethyl-vanillin or MA1 or MA2); 2.sup.nd: aqueous solution of NaOH 3M; 3.sup.rd: glutathione; 4.sup.th: Glycine; 5.sup.th: GST enzyme. Reaction is carried out at 25° C./220 rpm, during 5 days. Samples were subdued to HPLC analysis for chromatographic profile comparison (samples were collected at time 0 h, 16 h, 40 h, 66 h and 120 h).

[0322] HPLC characterization conditions:

[0323] (1) the mobile phase

[0324] Solution A: methanol;

[0325] Solution B: 1% Formic acid in ddH2O;

[0326] A:B ratio=35%:65%

[0327] (2) column: Agilent C18, 250 mm*4.6 mm

[0328] (3) Detection wavelength: 280 nm

[0329] (4) Column temperature: 25° C.

[0330] (5) Flow Rate: 1 ml/min

[0331] Results:

[0332] By HPLC chromatography, an evolution of the peaks was observed through time with both mixtures MA1 and MA2. Three major peaks at 2.7 min, 3.1 min and 3.5 min were observed at 0 hrs. The peak at 2.7 min increases with time demonstrating further degradation of the mixtures during enzymatic treatment with a GST.

[0333] It was supposed that the mechanism of action of the glutathione S-transferase was the cleavage of aryl ether bonds. It was confirmed by running the following model reaction with ethyl vanillin:

##STR00005##

[0334] Consumption of the ethyl-vanillin substrate was indeed observed by HPLC.

[0335] The step b) of enzymatic treatment with glutathione S-transferase led to partial oligomerization of the resin.

[0336] C—Step c) treating enzymatically by contacting mixture MA with a para-hydroxybenzoate hydroxylase

[0337] After performing step a) on resins R1 and R2 and obtaining respectively mixtures MA1 and MA2, step c) was performed according to the method below in order to evaluate the activity of Mutant enzyme issued from Pseudomonas aeruginosa: [0338] M010-2 (L199V and Y385F mutant enzyme issued from Pseudomonas aeruginosa), [0339] M012-2 (L199G, Y385F mutant enzyme issued from Pseudomonas aeruginosa), [0340] YM322-2 (L200V, Y385F, D39Y mutant enzyme issued from Corynebacterium glutamicum) and [0341] M020-1 (wild-type enzyme issued from Corynebacterium glutamicum).

[0342] Step a) Preparation of mixtures MA1 and MA2:

[0343] 1.sup.st Step: Prepare a solution of 1.98 g Resin+50.65 g deionized H.sub.2O; +90.00 g Na.sub.2HPO.sub.4.12H.sub.2O, +189 g deionized H.sub.2O, at 60° C. until complete solubilization of the Na.sub.2HPO.sub.4.12H.sub.2O salt;

[0344] 2.sup.nd Step: Evaporate at 80° C. under vacuum to remove water then add 300 g of deionized H.sub.2O, then evaporate again until complete removal of water.

[0345] 3.sup.rd Step: Repeat the 2.sup.nd step;

[0346] 4.sup.th Step: Add deionized H.sub.2O at 90° C. to the dry solid;

[0347] 5.sup.th Step: Add 9.96 g 13-Cyclodextrin to the previous solution (total weight 142 g), and mix;

[0348] 6.sup.th Step: Stir the mixture at 90° C. for two days;

[0349] 7.sup.th Step: Dilute the mixture to 200 ml;

[0350] The mixture obtained after performing step 1 to 7 on resin R1 is noted MA1.

[0351] The mixture obtained after performing step 1 to 7 on resin R2 is noted MA2.

TABLE-US-00002 TABLE B Vol. Glucose ET004 NADP FAD (4 g/l) PHBH ID Substrate Enzyme (ml) (g) (ml) (50 g/l) (ml) (μl) (ml) 1 MA2 None 10 1.875 0.6 0.5 25 0.00 2 MA2 M012-2 10 1.875 0.6 0.5 25 2.00 3 MA2 YM322-2 10 1.875 0.6 0.5 25 3.00 4 MA1 M020-1 10 1.875 0.6 0.5 25 1.75 5 MA1 YM322-2 19 1.875 0.6 0.5 25 3.00 6 MA1 M010-2 10 1.875 0.6 0.5 25 3.00

[0352] According to Table B, in a reactor of suitable volume, the components were added in the following order of 1.sup.st substrate (MA1 or MA2 obtained after step 7 of step a), 2.sup.nd Glucose, 3.sup.rd NADP, 4.sup.th FAD, 5.sup.th ET004 and 6.sup.th PHBH. Reaction is carried out at 35° C./220 rpm for 98 hours. Samples were subdued to HPLC analysis for chromatographic profile comparison (samples were collected at time 0 h, 30 h, 96 h).

[0353] HPLC assay conditions:

[0354] (1) the mobile phase

[0355] Solution A: methanol;

[0356] Solution B: 1% phosphoric acid in ddH.sub.2O;

[0357] A:B=30%:70%

[0358] (2) Column: Agilent C18, 250 mm*4.6 mm

[0359] (3) Detection wavelength: 210 nm

[0360] (4) Column temperature: 25° C.

[0361] (5) Flow Rate: 1 ml/min

[0362] Results:

[0363] By HPLC chromatography, an evolution of the peaks was observed through time with both mixtures MA1 and MA2 demonstrating further degradation of the mixtures during enzymatic treatment with a PHBH. In particular, the most interesting results were observed with enzymatic treatment of MA1 with M010-2 showing by HPLC a significant increase of peaks at 3.5 min and 4.2 min through time (see Tables 1 and 2)

TABLE-US-00003 TABLE 1 Resin R1 Peak area at 3.5 Peak area at 4.1 Peak area at 4.2 Time (hrs) min min min Strain M020-1 (ID-4) 0 103 — — 30 206 — — 96 958 937 150 Strain YM322-2 (ID-5) 0 330 — 372 96 489 — 575 Strain M010-2 (ID-6) 0 127 — 199 30 300 — 197 96 1011 — 1174

TABLE-US-00004 TABLE 2 Resin R2 Peak area at 3.5 Peak area at 4.1 Peak area at 4.2 Time (hrs) min min min None (ID-1) 0 — — — 30 — — — 96 182 — — Strain M012-2 (ID-2) 0 118 — — 30 229 — — 96 1071 2086 — Strain YM322-2 (ID-3) 0 325 — — 30 487 — — 96 1449 1253 405

[0364] It was supposed that the mechanism of action of the para-hydroxybenzoate hydroxylase

[0365] (PHBH) was the hydroxylation of aryl rings. It was confirmed by running the following model reactions:

[0366] a. Model reaction 1—with 3,4-dihydroxybenzoic acid as substrate:

##STR00006##

[0367] b. Model reaction 2—with Bisphenol A (BFA) as substrate:

##STR00007##

[0368] D—Enzymatic Treatment by Performing Concomitant Step b) and c)

[0369] Step a):

[0370] (1) Prepare a solution of 1.98 g Resin+50.65 g deionized H.sub.2O; +90.00 g Na.sub.2HPO.sub.4.12H.sub.2O, +189 g deionized H.sub.2O are mixed at 60° C. until complete solubilization of the Na.sub.2HPO.sub.4.12H.sub.2O salt;

[0371] (2) Evaporate the solution from the previous step at 80° C. under vacuum to remove the water, then add about 300 g of deionized H.sub.2O, then evaporate again until complete removal of water,

[0372] (3) Repeat the 2nd step again;

[0373] (4) Add deionized H.sub.2O at 90° C. to the dry solid obtained after step 3;

[0374] (5) Add 9.96 g β-Cyclodextrin to the mixture obtained after step 4 (total weight 142 g), and mix;

[0375] (6) Stir the mixture at 90° C. for two days;

[0376] (7) Dilute the mixture to obtain a volume of 200 ml;

[0377] The mixture obtained after performing step 1 to 7 on resin R1 is noted MA1.

[0378] The mixture obtained after performing step 1 to 7 on resin R2 is noted MA2.

[0379] Steps b) and c):

TABLE-US-00005 TABLE C Weight Of sub- NaOH Glu- NADP FAD Sub- strate solution cose GSH Glycine ET004 (20 g/l) (4 g/l) PHBH GST ID strate (g) (ml) (g) (g) (g) (ml) (ml) (μl) (ml) (ml) 1 MA2 30 51.7 11.25 0.22 1.07 3.6 3 150 10 7 2 MA1 30 50 11.25 0.22 1.07 3.6 3 150  9 7 3 MA2 30 68.7 11.25 0.22 1.07 3 3 150  0 0 4 MA1 30 68.7 11.25 0.22 1.07 3 3 150  0 0

[0380] According to table C, the compounds were added in a suitable reactor in the following order of: 1.sup.st substrate (mixture MA1 or MA2), 2.sup.nd aqueous solution of NaOH (pH9), 3.sup.rd Glucose, 4.sup.th reduced glutathione (GSH), 5.sup.th Glycine, 6.sup.th NADP, 7.sup.th FAD, 8.sup.th ET004, 9.sup.th PHBH and 10.sup.th GST. The reaction medium is stirred until reach homogeneity. The reaction is conducted at 30° C./220 rpm for 72-96 h. The reaction medium was sampled at 0 h, 20 h, 48 h, 72 h and 96 h. Control experiments were also conducted in which no enzyme was used.

[0381] The mixture obtained after performing step b) and c) on MA1 is noted MC1.

[0382] The mixture obtained after performing step b) and c) on MA2 is noted MC2.

[0383] The mixture obtained after conducting the experiment with no enzyme on MA1 is noted MC1-CTRL.

[0384] The mixture obtained after conducting the experiment with no enzyme on MA2 is noted MC2-CTRL.

[0385] HPLC assay condition

[0386] (1) the mobile phase

[0387] Solution A: methanol;

[0388] Solution B: 1% phosphoric acid in ddH2O;

[0389] A:B=30%:70%

[0390] (2) column: Agilent C18, 250 mm*4.6 mm

[0391] (3) Detection wavelength: 210 nm

[0392] (4) Column temperature: 25° C.

[0393] (5) Flow Rate: 1 ml/min

TABLE-US-00006 TABLE 3 Time Peak area at Peak area at Peak area at (hrs) 2.7 min 3.5 min 4.2 min MC1 0 — 101 0 20 — 262 108 72 — 1148 105 96 — 1927 158 MC1-CTRL 0 — — — 24 — 145 — 48 — 200 — 72 — 262 — MC2 0 942 421 — 48 1339 606 — 72 1374 1174 385 96 1405 1241 283 MC2-CTRL 0 — — — 24 — 210 — 48 — 118 — 72 — 245 —

[0394] Table 3 above shows the influence of the presence of the enzymes GST and PHBH and their effect when used concomitantly. The evolution of the area of the peaks according to time shows an enrichment of molecular species that elude or coelude in specific retention times for mixtures MC1 and MC2 derived from resins R1 and R2 and treated with GST and PHBH. To the contrary, barely nothing happened with samples of resins R1 and R2 treated with no enzyme (MC1-CTRL and M2-CTRL).

[0395] E—Scale-Up

[0396] Step a)

[0397] 1.sup.st Step: Prepare a solution of 10.04 g Resin R1+102.08 g deionized H.sub.2O; +100.01 g Na.sub.2HPO.sub.4.12H.sub.2O, mix at 60° C. until complete solubilization of the Na.sub.2HPO.sub.4.12H.sub.2O salt;

[0398] 2.sup.nd Step: Evaporate the solution from the previous step at 80° C. under vacuum to remove water, then add about 300 g of deionized H.sub.2O, then evaporate again until complete removal of water;

[0399] 3.sup.rd Step: Repeat the 2nd step;

[0400] 4.sup.th Step: Add deionized water at 90° C. to the dry solid obtained after step 3;

[0401] 5.sup.th Step: Add 50.72 g 3-Cyclodextrin to the previous solution, and mix;

[0402] 6.sup.th Step: Stir the mixture at 90° C. for two days;

[0403] 7.sup.th Step: Dilute the mixture to obtain a volume of about 1200 ml in the reactor;

[0404] The broth obtained after the material dilution at the 7.sup.th Step is actually used to explore the degradation of resin R2 and R2.

[0405] The mixture obtained after performing step 1 to 7 on resin R1 is noted MA1. The mixture obtained after performing step 1 to 7 on resin R2 is noted MA2. Steps b) and c)

TABLE-US-00007 TABLE D Weight of Glu- NADP FAD NaOH substrate cose GSH Glycine ET004 (20 g/l) (4 g/l) PHBH GST 3M N. Substrate (g) (g) (g) (g) (ml) (ml) (ml) (ml) (ml) (ml) 1 MA1 1200 187 0.1 17.8 10 12.5 2.5 80 33 662 2 MA2 1200 187 0.1 17.8 10 12.5 2.5 80 33 662

[0406] According to table D, the compounds were added in a 5 L reactor in the following order of: 1.sup.st substrate (MA1 or MA2), 2.sup.nd NaOH 3M, 3.sup.rd Glucose, 4.sup.th GSH, 5.sup.th Glycine, 6.sup.th NADP, 7.sup.th FAD, 8.sup.th ET004, 9.sup.th PHBH and 10.sup.th GST. The reaction medium is stirred at 200 rpm until homogeneity is reached. Compressed air was pumped at a rate of 1 L/min in the reactor. The reaction is conducted at 30° C./200 rpm for 66 h. The mixture obtained was filtrated and dried.

[0407] Starting from MA1, the dried mixture obtained is noted MC1.

[0408] Starting from MA2, the dried mixture obtained is noted MC2.

[0409] The mixture obtained after conducting the experiment with no enzyme on MA1 is noted MC1-CTRL.

[0410] The mixture obtained after conducting the experiment with no enzyme on MA2 is noted MC2-CTRL.

TABLE-US-00008 TABLE 4 Resin R1 Time and temperature for Weight MC1- Weight drying the resulting mixture CTRL (g) MC1 (g) 0 hrs 9.85 10.04 3 hrs at 90° C. 9.77 7.98 5 hrs at 90° C. 9.75 7.96

TABLE-US-00009 TABLE 5 Resin R2 Time and temperature for Weight MC2- Weight drying the resulting mixture CTRL (g) MC2 (g) 0 hrs 9.76 10.02 3 hrs at 90° C. 9.69 8.64 5 hrs at 90° C. 9.67 8.62

[0411] Tables 4 and 5 show that when both Resins R1 and R2 treated with GST and PHBH, a loss of material is observed.

[0412] Step d)

[0413] The dried mixtures MC1 and MC2 were sampled (around 1 g of mass) and treated with a 2M NaOH aqueous solution at 90° C. under stirring for 96 h (4 days). The mixtures were then filtrated, washed and dried at 90° C. for 5 hrs, and the loss of mass registered in Table 6 below.

[0414] Starting from MC1, the dried mixture obtained is noted MD1.

[0415] Starting from MC2, the dried mixture obtained is noted MD2.

[0416] The mixture obtained after conducting the experiment with no enzyme on MA1 is noted MD1-CTRL.

[0417] The mixture obtained after conducting the experiment with no enzyme on MA2 is noted MD2-CTRL.

TABLE-US-00010 TABLE 6 mass after NaOH mass before treatment and NaOH drying at 90° C. treatment (g) for 5 hrs (g) MD1-CTRL 1.01 1.0 MD1 1.02 0.9 MD2-CTRL 1.00 0.96 MD2 1.00 0.86

[0418] Resins R1 and R2 chemoenzymatically treated are more sensitive to the action of NaOH, losing higher amounts of mass, compared to untreated resins R1 and R2. The chemoenzymatic treatment was responsible for enhancing the solubility of portions of R1 and R2 in alkaline media.

[0419] F—Two Rounds of the Method According to the Invention

[0420] 1.sup.st Round:

[0421] step a)

[0422] (1) Prepare a solution of 1.98 g Resin R2+50.65 g ddH2O; +90.00 g Na.sub.2HPO.sub.4.12H.sub.2O, +189 g ddH.sub.2O, mix at 60° C. until complete solubilization of the Na.sub.2HPO.sub.4.12H.sub.2O salt;

[0423] (2) evaporate at 80° C. under vacuum to remove the water, then add about 300 g of deionized water, then evaporate again until complete removal of water;

[0424] (3) Repeat the 2nd step;

[0425] (4) Add deionized water at 90° C. to the dry solid;

[0426] (5) Add 9.96 g β-Cyclodextrin to the previous solution (total weight 142 g), and mix;

[0427] (6) Stir the mixture at 90° C. for two days;

[0428] (7) Diluted the mixture to 200 ml;

[0429] (8) Resin R1 was dealt with the same process of (1)-(7).

[0430] The mixture obtained after performing step 1 to 7 on resin R1 is noted MA1. The mixture obtained after performing step 1 to 7 on resin R2 is noted MA2.

[0431] Steps b) and c)

TABLE-US-00011 TABLE E Weight of NaOH Gly- NADP FAD Sub- substrate 3M Glucose GSH cine ET004 (20 g/l) (4 g/l) PHBH GST ID strate (g) (ml) (g) (g) (g) (ml) (ml) (μl) (ml) (ml) 1 MA2 30 51.7 11.25 0.22 1.07 3.6 3 150 10 7 2 MA1 30 50 11.25 0.22 1.07 3.6 3 150  9 7

[0432] According to table E, the compounds were added in a suitable reactor in the following order of: 1.sup.st substrate (MA1 or MA2), 2.sup.nd NaOH 3M, 3.sup.rd Glucose, 4.sup.th GSH (substrate for GST enzyme), 5.sup.th Glycine, 6.sup.th NADP (cofactor), 7.sup.th FAD (cofactor), 8.sup.th ET004 (enzyme), 9.sup.th PHBH (enzyme) and 10.sup.th GST (enzyme). The reaction medium is stirred until reach homogeneity. The reaction is conducted at 30° C./220 rpm for 72-96 h. [0433] (1) The resulting mixtures were left to decant and the supernatant was removed. [0434] (2) Water was added, left to decant again, then the supernatant was removed. [0435] (3) Step (2) was repeated until the supernatant was clear. [0436] (4) The supernatant was removed.

[0437] Starting from MA1, the mixture obtained is noted MC1.

[0438] Starting from MA2, the mixture obtained is noted MC2.

[0439] Step d)

[0440] (1) Add 50 ml of 2 M NaOH solution to mixtures MC1 or MC2;

[0441] (2) Stir the mixture at 90° C. for 4 days;

[0442] (3) Remove the supernatant from the mixture and wash the solid three times with water.

[0443] Starting from MC1, the mixture obtained is noted MD1.

[0444] Starting from MC2, the mixture obtained is noted MD2.

[0445] Control experiments without enzymes PHBH and GST were also conducted.

[0446] 2.sup.nd Round:

[0447] Enzymatic Treatment in 2.sup.nd Round (Steps b and c):

[0448] The solid from 1.sup.st Round is mixed again with the other chemicals and enzymes in the following order: 1.sup.st substrate (MD1 from 1.sup.st Round), 2.sup.nd NaOH 3M, 3.sup.rd Glucose, 4.sup.th GSH, 5.sup.th Glycine, 6.sup.th NADP, 7.sup.th FAD, 8.sup.th ET004, 9.sup.th PHBH and 10.sup.th GST. The reaction medium is stirred until reach homogeneity. The reaction is carried out at 30° C., 220 rpm for 120 h (5 days).

[0449] (1) The resulting mixture was left to decant and the supernatant was removed.

[0450] (2) Water was added, left to decant again, then the supernatant was removed.

[0451] (3) Step (2) was repeated until the supernatant was clear.

[0452] (4) The supernatant was removed.

[0453] Starting from MD1, the mixture obtained is noted MC1′.

[0454] NaOH Treatment in the 2.sup.nd Round (Step d):

[0455] (1) Add 50 ml of 2 M NaOH solution to MC1′;

[0456] (2) Stir the mixture at 90° C. for 4 days;

[0457] (3) Remove the supernatant from the mixture and wash the solid three times with water;

[0458] (4) Dry the solid at 70° C.

[0459] Starting from MC1′, the mixture obtained is noted MD1′.

TABLE-US-00012 TABLE 7 Weight loss Control R1  2% R1-after 1.sup.st round 31% R1-after 2.sup.nd round 53% Control R2  4% R2-after 1.sup.st round 24%