Process for the preparation of ammeline

Abstract

The present invention relates to a new process for the preparation of ammeline and/or ammelide from melamine by a solid-to-solid reaction in an aqueous reaction mixture comprising a biocatalyst, wherein the biocatalyst comprises at least one enzyme belonging to the amidohydrolase superfamily and having aminohydrolase activity towards 1,3,5-triazine compounds. The invention further relates a product obtainable by the process according to the invention, wherein the product comprises ammeline and/or ammelide.

Claims

1. A process for the preparation of ammeline and optionally ammelide from melamine comprising: a) contacting solid melamine in an aqueous reaction mixture with a biocatalyst; b) incubating the aqueous reaction mixture comprising solid melamine and the biocatalyst for sufficient time to convert the melamine into a product comprising solid ammeline: and c) recovering the product, wherein the biocatalyst comprises at least one enzyme belonging to the amidohydrolase superfamily and having amidohydrolase activity towards 1, 3, 5-triazine compounds, and wherein the at least one enzyme comprises an amino acid sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

2. The process according to claim 1, wherein the enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

3. The process according to claim 1, wherein the melamine is present in a loading of at least 1.0 mass %, relative to the total mass of the aqueous reaction mixture before the contacting with the biocatalyst.

4. The process according to claim 3, wherein the melamine is present in a loading of at least 10 mass %, relative to the total mass of the aqueous reaction mixture.

5. The process according to claim 4, wherein the melamine is present in a loading of at least 20 mass %, relative to the total mass of the aqueous reaction mixture.

6. The process according to claim 1, wherein the melamine is converted into ammeline, and optionally ammelide, at a pH selected between 5 and 10.

7. The process according to claim 6, wherein the melamine is converted into ammeline, and optionally ammelide, at a pH selected between 6.5 and 7.5.

8. The process according to claim 6, wherein the melamine is converted into ammeline, and optionally ammelide, at a pH selected below 6.5.

9. The process according to claim 6, wherein the melamine is converted into ammeline, and optionally ammelide, at a pH selected above 7.5.

10. The process according to claim 1, wherein the aqueous reaction mixture comprises an organic solvent.

11. The process according to claim 1, wherein at least about 96% of the melamine is converted into ammeline and ammelide.

12. The process according to claim 1, wherein the process produces substantially no cyanuric acid.

13. The process according to claim 1, wherein the product comprises at least 95 mass % of ammeline and ammelide and no more than 5 mass % of melamine.

Description

EXAMPLES

(1) Materials and General Methods

(2) 1. Melamine Substrate; Reference Materials Ammeline, Ammelide and Cyanuric Acid

(3) Melamine (from OCl-Nitrogen), with a chemical purity of >99.9% was applied for the examples. Ammeline (from Hicol) with a chemical purity of 97.5% was used as reference material in HPLC analyses. Ammelide with a chemical purity of 99.7% was used as reference material in HPLC analyses. Cyanuric acid with a chemical purity of >99% was used as reference material in HPLC analyses.
2. Preparation of Biocatalysts
2.a. Cloning and Expression of Recombinant Enzymes

(4) Five genes from different organisms encoding enzymes with aminohydrolase activity towards 1,3,5-triazine compounds were selected to exemplify the invention; namely SEQ ID NO: 10 (gi_11890745), SEQ ID NO: 11 (gi_310815990), SEQ ID NO: 12 (gi_4033703), SEQ ID NO: 13 (gi_27378991) and SEQ ID NO: 14 (gi_309703244) encoding SEQ ID NO: 5 (AAG41202.1), SEQ ID NO: 6 (YP_003963954.1), SEQ ID NO: 7 (Q52725.2), SEQ ID NO: 8 (NP_770520.1) and SEQ ID NO: 9 (CBJ02579.1), respectively (Table 1).

(5) TABLE-US-00001 TABLE 1 Overview of enzymes, donor organisms, accession numbers and recombinant vectors. Codon Enzyme Gene pair SEQ ID NO SEQ ID NO optimized (accession (accession gene.sup.(*.sup.) Recombinant Enzyme number) number) SEQ ID NO vectors Melamine 5 10 1 pBAD_Meldeam_Aci deaminase from (AAG41202.1) (gi_11890745) Acidovorax citrulli NRRL B-12227 Melamine 6 11 2 pBAD_Meldeam_Kvu deaminase from (YP_003963954.1) (gi_310815990) Ketogulonicigenium vulgare Y25 s-Triazine 7 12 3 pBAD_TrzA_Rco hydrolase from (Q52725.2) (gi_4033703) Rhodococcus corallinus NRRL B-15444R Guanine 8 13 4 pBAD_Guadeam_Bja deaminasefrom (NP_770520.1) (gi_27378991) Bradyrhizobium japonicum USDA 110 Guanine 9 14 pBAD_Guadeam_Eco deaminase from (CBJ02579.1) (gi_309703244) Escherichia coli ETEC H10407 .sup.(*.sup.) For expression in Escherichia coli

(6) Four out of the five target genes, i.e. SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, were codon pair optimized for expression in Escherichia coli, resulting in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively (sequence listing). Codon pair optimization was performed according to a procedure described in WO2008/000632.

(7) For larger scale expression experiments, E. coli RV311 cells were made chemically competent using the Z-Competent E. coli Transformation Kit (Zymo Research, Irvine, Calif., USA) and transformed with the pBAD recombinant vectors isolated as described above from the E. coli TOP10 strains. Transformants were selected on LB agar plates containing 100 g/ml neomycin. From these plates 50 ml precultures in LB medium containing 50 g/ml neomycin were inoculated and incubated on an orbital shaker at 180 rpm and 28 C. After overnight incubation the precultures were used to inoculate 1 l LB expression cultures (in 2 l baffled Erlenmeyer flasks) to starting OD.sub.620s of 0.05. These expression cultures were incubated on an orbital shaker at 180 rpm and 28 C. In one third of the cultures, the melamine deaminase gene expression was induced after 8 h by addition of 0.02% (w/v) L-arabinose and incubation was continued overnight. Subsequently these cultures were harvested by centrifugation at 10,000 g for 10 min. The other two third of the cultures were also incubated overnight and induced as described above after approximately 24 h. After 4 and 8 h, respectively, also these cultures were harvested by centrifugation.

(8) 2.b. Fermentation of Biocatalyst on 10 l Scale

(9) Fermentation of E. coli RV311 expressing the pBAD recombinant vectors was performed in a fed-batch fermentation on 10 l scale, using glucose as a carbon source.

(10) 2.c. Preparation of Cell Free Extract on Small Scale (1-10 ml)

(11) The cell pellets were resuspended in twice the volume of their wet weight with ice-cold 100 mM potassium phosphate buffer pH 7.0. Cell-free extracts (CFEs) were obtained by sonification of the cell suspensions using a Sonics (Meyrin/Satigny, Switzerland) Vibra-Cell VCX130 sonifier (output 100%, 10 sec. on/10 sec. off, for 10 min) with cooling in an ice/acetone bath and subsequent centrifugation in an Eppendorf 5415R centrifuge (Hamburg, Germany) at 13,000g and 4 C. for 30 min. The supernatants (=CFEs) were transferred to fresh tubes and stored on ice for immediate use or stored at 20 C.

(12) 2.d. Preparation of Cell Free Extract on Larger Scale (10-250 ml Scale)

(13) To 76.3 g wet cells (frozen), 152.6 g potassium phosphate buffer (100 mM pH=7) was added. Cells were suspended and put on ice. Subsequently, 1125 mg lysozym and 50 l benzonase were added, mixed and put at 20 C. overnight. The next morning the suspension was put at 37 C. room on a shaker for 2.25 h. The suspension was cooled afterwards on ice and subsequently divided over 8 tubes of 50 ml (28 ml each) and each suspension was sonicated for 2 min, using a Sonics (Meyrin/Satigny, Switzerland) Vibra-Cell VCX130 sonifier (output 100%, 10 sec. on/10 sec. off, for 10 min) with cooling in an ice/acetone bath. Subsequently, the suspensions were stored on ice at 4 C. room for 4 h. Next, the suspensions were centrifuged at 15.000 rpm for 15 min. The obtained supernatants were collected and stored at 20 C.

(14) 3. Protein Determination

(15) The protein concentrations in the CFEs were determined using a modified protein-dye binding method as described by Bradford in Anal. Biochem. 72: 248-254 (1976). Of each sample 50 l in an appropriate dilution was incubated with 950 l reagent (100 mg Brilliant Blue G250 dissolved in 46 ml ethanol and 100 ml 85% ortho-phosphoric acid, filled up to 1,000 ml with Milli-Q water) for at least five minutes at room temperature. The absorption of each sample at a wavelength of 595 nm was measured in a PerkinElmer Lambda35 UV/VIS spectrophotometer. Using a calibration line determined with solutions containing known concentrations of bovine serum albumin (BSA, ranging from 0.0125 mg/ml to 0.20 mg/ml), the protein concentration in the samples was calculated.

(16) 4. SDS-PAGE Analysis

(17) The recombinant expression in E. coli was analyzed by SDS-PAGE of the E. coli TOP10 CFEs and of the E. coli RV 311 CFEs and compared to a CFE with an overexpressed control protein.

(18) 5. Conversion Reactions and Product Isolation

(19) 5.a. Conversion ReactionsGeneral Description

(20) Conversion reactions were either performed in a glass reactor of 80 ml containing a propeller stirrer or in a glass reactor of 1600 ml, containing a turbine stirrer and pH-stat equipment (type 718 Stat Titrino from Metrohm). A stirrer speed of about 500 rpm was applied.

(21) In general, a conversion was performed as follows: a certain amount of melamine was added to a buffer (100 mM K.sub.2HPO.sub.4/1 mM MgSO.sub.4.7H.sub.2O) at 37 C. to reach a desired loading. The pH was adjusted to the desired value, using 1M H.sub.3PO.sub.4 (for obtaining a pH of 7 or lower) or 1M NaOH (for obtaining a pH of 8 or higher). Subsequently, the biocatalyst was added to start the conversion. Samples were taken in the course of the conversion for HPLC analysis as described in paragraph 6.

(22) 5.b. Product Isolation

(23) Product isolation was performed after a certain reaction time by pouring the reaction mixture over a P3 glass filter, using vacuum (about 750 mbar). The filter cake was washed three times with water. The obtained product was dried overnight.

(24) 6. HPLC Analysis

(25) 6.a. Sample Preparation for HPLC-Analysis

(26) Samples of the aqueous reaction mixtures or samples of the isolated products were diluted in first instance with formic acid to a total 1,3,5-triazine compounds concentration of about 0.5 mass % and subsequently diluted 50 times with water before subjecting to HPLC analysis.

(27) 6.b. HPLC Analysis Method

(28) Two 250 mm Prevail C18 columns are used. The critical separation takes place at 0% acetonitrile. The columns are to be equilibrated for at least 8 minutes after the gradient.

(29) The specific analytical conditions on the HPLC used are:

(30) Columns: Prevail C18 2 (250 mm4.6 mm ID5 m) Eluent A: HClO.sub.4 pH=2.0 (1.63 g 70% HClO.sub.4/l water) Eluent B: Acetonitrile Flow: 1.2 ml/min Injection volume: 5 l Column temperature: 15 C. Detection wavelength: 195 nm

(31) TABLE-US-00002 Time (min) % B 0 0 12 0 12.5 80 13.5 80 14 0 22 Stop

Example 1: Preparation of Ammeline and/or Ammelide from Melamine by a Solid-to-Solid Reaction in an Aqueous Reaction Mixture

(32) Reactions were performed in 80 ml reactors with a filling volume of about 55 ml, containing a stirrer and pH-stat equipment.

(33) For the test reaction, 555 mg of melamine was added to 47 g buffer (100 mM K.sub.2HPO.sub.4/1 mM MgSO.sub.4.7H.sub.2O) at 37 C. The pH was adjusted to pH 9.5 with 1.65 g 1M NaOH. Subsequently, 0.25 ml of cell free extract of E. coli RV311 pBAD_Meldeam_Aci was added to start the reaction, thereby obtaining a final concentration of cell free extract of 0.5 vol %. The melamine loading was 1.1 mass %. The pH was kept constant at 9.5 by titration with 1 M NaOH.

(34) A chemical blank reaction was run in parallel, using the same procedure as described above, with the exception that no biocatalyst was added. A biological blank reaction was run in parallel, using the same procedure as described above, with the exception that cell free extract is obtained from E. coli RV311 harboring a pBAD recombinant vector having a gene insert that encodes an enzyme not able to convert melamine (in this case a P450 monooxygenase enzyme from Bacillus megaterium BM3).

(35) After 18 h reaction time, samples were taken from the three reaction mixtures for HPLC analysis. Results are shown in Table 2.

(36) TABLE-US-00003 TABLE 2 HPLC analyses of the reaction mixtures after 18 h reaction time Cyanuric Reaction Melamine Ammeline Ammelide acid Conversion mixture (mol %) (mol %) (mol %) (mol %) (%) Test 1.2 97.7 1.1 0 98.8 reaction Chemical 99.8 0.2 0.0 0 0.2 blank Biological 99.9 0.1 0.0 0 0.1 blank

(37) The results in Table 2 show that the conversion of melamine to ammeline and ammelide is due to the activity of the biocatalyst (i.e. cell free extract of E. coli RV311 pBAD_Meldeam_Aci). The melamine initially added to the reaction mixture is almost fully converted to ammeline and ammelide within 18 h. No cyanuric acid was formed. No significant conversion of melamine took place in the chemical blank reaction mixture, showing that the melamine is chemically stable during the reaction time, under the conditions applied. Furthermore, no significant conversion of melamine took place in the biological blank reaction mixture.

Example 2: Effect of pH on the Ammeline:Ammelide Ratio

(38) Conversion reactions were performed in 80 ml reactors with a filling volume of about 55 ml containing a stirrer and pH-stat equipment.

(39) 555 mg of melamine was added to 47 g buffer (100 mM K.sub.2HPO.sub.4/1 mM MgSO.sub.4.7H.sub.2O) at 37 C. The pH was adjusted to the desired value, using 1M H.sub.3PO.sub.4 (for obtaining a pH of 7 or lower) or 1M NaOH (for obtaining a pH of 8 or higher). Subsequently, 0.25 ml of cell free extract of E. coli RV311 pBAD_Meldeam_Aci was added to start the reaction, thereby obtaining a final concentration of cell free extract of 0.5 vol %. The melamine loading was 1.1 mass %. The pH was kept constant by titration with 1 M H.sub.3PO.sub.4 or 1 M NaOH. Samples were taken over time for HPLC analysis. At maximum conversion, the solid products were isolated and analyzed as described in materials and general methods. Results are shown in Table 3.

(40) TABLE-US-00004 TABLE 3 Maximum conversion and analysis results of the isolated solid products Ammeline:ammelide Melamine Ammeline Ammelide ratio at maximum Conversion pH (mass %) (mass %) (mass %) conversion (%) 5 3.5 91.5 5.0 18.3 96.1 6 3.2 86.9 9.9 8.8 96.8 7 3.6 75.2 21.2 3.5 96.4 8 3.7 90.0 6.3 14.3 96.3 9 1.8 96.5 1.7 56.8 98.2 9.5 1.2 97.9 0.9 108.8 98.8 10 1.0 98.4 0.6 164 99.0

(41) The results in Table 3 show that the ammeline:ammelide ratio obtained in the reaction mixture can be fine-tuned by the pH. Within a pH range of 7 to 10, the higher the pH, the higher the ammeline:ammelide ratio. An inverse trend was observed at pH values below 7, wherein a higher pH resulted in a lower ammeline:ammelide ratio. It is noteworthy to mention that cyanuric acid was never detected.

Example 3: Effect of Melamine Loading on the Ammeline:Ammelide Ratio

(42) Conversion reactions were performed in 80 ml reactors with a filling volume of about 55 ml containing a stirrer and pH-stat equipment.

(43) Melamine was added to 47 g buffer (100 mM K.sub.2HPO.sub.4/1 mM MgSO.sub.4.7H.sub.2O) at 37 C. to a loading of 1.1 mass %, 9 mass % or 17.5 mass %. The pH was adjusted to 9.5 using 1 M NaOH. Subsequently, to start the reactions, cell free extract of E. coli RV311 pBAD_Meldeam_Aci was added to a concentration of 0.5 vol %, 5 vol % or 10 vol %, respectively. The pH was kept constant during the conversion by titration with 1 M NaOH. Samples were taken over time for HPLC analysis. At maximum conversion, the solid products were isolated and analyzed as described in materials and general methods. Results are shown in Table 4.

(44) TABLE-US-00005 TABLE 4 Maximum conversion and analysis results of the isolated solid products Melamine Ammeline:ammelide loading Melamine Ammeline Ammelide ratio at maximum Conversion (mass %) (mass %) (mass %) (mass %) conversion (%) 1.1 1.2 97.9 0.9 108.8 98.8 9 0.8 98.9 0.3 329.7 99.2 17.5 1.0 98.8 0.2 494.0 99.0

(45) The results in Table 4 show that at the chosen reaction conditions, the ammeline:ammeline ratio can be fine-tuned by adjusting the melamine loading; the higher the melamine loading under the chosen reaction conditions, the higher the ammeline:ammelide ratio. It is noteworthy to mention that cyanuric acid was never detected.

Example 4: Preparation of Ammeline and/or Ammelide by a Solid-to-Solid Reaction in an Aqueous Mixture on 1 l Scale

(46) A reaction mixture was prepared in a stirred 1.6 l glass reactor, by adding 125.2 g melamine to 1050 ml buffer (100 mM K.sub.2HPO.sub.4/1 mM MgSO.sub.4.7H.sub.2O). The melamine solid substrate was stirred for 20 min at 37 C. after which the pH was adjusted to pH 9.5 by adding 0.05 g 5M NaOH. To start the reaction 62.5 ml of cell free extract of E. coli RV311 pBAD_Meldeam_Aci was added (5 vol %), after which the pH was adjusted to pH 9.5 by adding another 0.1 g of 5 M NaOH. The melamine loading was 10.1 mass %. During the conversion, the pH was kept constant at pH 9.5 by titrating a solution of 1M H.sub.3PO.sub.4 applying a pH-stat equipment. Samples were taken for HPLC analysis. After 5 h, the reaction was stopped and the solid product was isolated and analyzed as described in materials and general methods. A total of 125 g of product was obtained, containing 98.6 mass % ammeline, 0.4 mass % ammelide and 1 mass % melamine, corresponding to a conversion of 99%. No cyanuric acid was formed.