A fermentation process for the preparation of xanthine or xanthine derivatives

Abstract

A fermentation process for the preparation of xanthine and xanthine derivatives. The disclosure generally relates to a fermentation process for the preparation of xanthine or xanthine derivatives such as 7-methylxanthine (7-MX), theobromine, theophylline, 3-methylxanthine or mixtures thereof. The process results in improved yield and increased efficiency on industrial scale compared to batch process of preparation.

Description

[0001] TITLE

[0002] A fermentation process for the preparation of Xanthine or Xanthine derivatives

[0003] TECHNICAL FIELD

[0004] The disclosure generally relates to a fermentation process for the preparation of xanthine or xanthine derivatives such as 7-methylxanthine (7-MX), theobromine, theophylline, 3- methylxanthine.

[0005] BACKGROUND

[0006] Methylxanthine A-demethylase (Ndm) possesses broad substrate specificity. Methylxanthine A-demethylase (Ndm) purified from CBB5 was disclosed in W02013059507A1. This was characterized as a soluble enzyme composed of two enzyme subunits, NdmA and NdmB, with apparent molecular mass of 40 and 35 kDa, respectively. The A -dem ethylation activity of Ndm, the oxygenase component, was dependent on a specific electron carrier reductase present in CBB5, NAD(P)H and oxygen.

[0007] Caffeine (1,3,7-trimethylxanthine) and related TV-methylated xanthines are purine alkaloids that are extensively used as psychoactive substances and food ingredients by humans.

[0008] W02013059507A1 discloses isolated nucleic acid encoding one or more gene products that allow for degradation of caffeine and related structures, and for preparation of intermediates in that catabolic pathway.

[0009] W02013059507A1 discloses the utilization of caffeine by CBB5 likely occurs via N- demethylation in a preferential sequence. The AT - ethyl group is first removed from caffeine by NdmA, forming theobromine, which is the preferred substrate of NdmB. NdmB then removes the AT-methyl group, producing 7-methylxanthine. Based on activity in partially purified fraction as well as gene-annotation, NdmC is proposed to catalyze A-demethylation. This ordered N- demethylation of caffeine is supported by the catalytic efficiencies of NdmA and NdmB on various methylxanthines. Both NdmA and NdmB are monooxygenases; one oxygen is consumed per N- methyl group removed as formaldehyde. NdmD appears to be the sole reductase for transfer electrons from NADH to NdmA, NdmB and possibly NdmC for oxygen activation and N- demethylation to formaldehyde.

[0010] W02013059507A1 discloses that to produce alkylxanthines, NdmA is introduced to host cells, e.g., Pichia cells which may be spray-dried. Sources of caffeine or theophylline are mixed with spray-dried cells and the redox reactions yield products such as theobromine and 3- methylxanthine respectively. NdmB is introduced to host cells, e.g., Pichia cells which may be spray-dried. Sources of caffeine, theobromine or theophylline are mixed with the cells and the redox reactions yield products 1,7-dimethylxanthine, 7-methylxanthine and 1 -methylxanthine respectively.

[0011] Alternatively, NdmA, NdmB and NdmD are introduced to Pichia cells which are spray- dried, and caffeine or theobromine is added. Redox reactions with those cells, e.g., spray-dried cells, yield products 7-methylxanthine. NdmC is introduced to host cells such as Pichia cells, which may be spray-dried, and caffeine or theobromine is added to these cells. Redox reactions in the presence of the cells provide products 1,3-dimethylxanthine and / or 3 -methylxanthine.

[0012] NdmA, B, C and D are introduced to host cells such as Pichia cells which may be spray- dried, and a source of caffeine, theobromine or theophylline is added to those cells. Redox reactions result in products including a variety of di- and mono-methylxanthines.

[0013] It is observed that the prior art discloses resting cell processes for the preparation of demethylated products wherein the yield is limited. It results in increased cost and reduced efficiency on industrial scale.

[0014] SUMMARY OF THE INVENTION

[0015] In one embodiment the specification discloses a fermentation process for the preparation of xanthine or xanthine derivatives comprising;

[0016] (a) inserting genes selected from NdmA, NdmB, NdmC, NdmD or mixtures thereof in a suitable host,

[0017] (b) fermenting in a suitable media for a suitable time, to obtain a fermentation broth,

[0018] (c) optionally, centrifuging and separating the cells, followed by suspension in a buffer with pH between 6 to 8 under optimal temperature conditions,

[0019] (d) adding one lot of a substrate xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl; provided that at least one is methyl,

[0020] (e) optionally, checking the consumption of the substrate,

[0021] (f) adding another and further lots of the substrate after every 2 to 8 hours,

[0022] (g) isolating a demethylated product xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl, provided that all of the R1, R2and R3are not methyl at the same time.

[0023] In another embodiment one isolation of product is performed after addition of at least 40 lots of substrates, another isolation of product is performed after addition of at least 20 lots of substrates and further isolation of product is performed after addition of at least 10 lots of substrates.

[0024] In another embodiment the gene in step (a) is mixture of NdmA, NdmB and NdmD; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 7-methylxanthine (7 -MX).

[0025] In another embodiment the gene in step (a) is NdmA; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is theobromine or wherein the gene in step (a) is NdmA; the substrate xanthine derivative in step (d) is theophylline and the demethylated product xanthine derivative in step (g) is 3 -methylxanthine.

[0026] In another embodiment the gene in step (a) is NdmB; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 1,7- dimethylxanthine, or, wherein the gene in step (a) is NdmB; the substrate xanthine derivative in step (d) is theobromine and the demethylated product xanthine derivative in step (g) is 7-methylxanthine, or, wherein the gene in step (a) is NdmB; the substrate xanthine derivative in step (d) is theophylline and the demethylated product xanthine derivative in step (g) is 1 -methylxanthine. In another embodiment the gene in step (a) is mixture of NdmA and NdmB; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 7-methylxanthine, or, wherein the gene in step (a) is mixture of NdmA and NdmB; the substrate xanthine derivative in step (d) is mixture of caffeine and theobromine and the demethylated product xanthine derivative in step (g) is 7-methylxanthine.

[0027] In another embodiment the gene in step (a) is NdmC; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 1,3- dimethylxanthine, or, wherein the gene in step (a) is NdmC; the substrate xanthine derivative in step (d) is theobromine and the demethylated product xanthine derivative in step (g) is 3 -methylxanthine.

[0028] All the above embodiments can alternatively be carried out in resting cell process, where in

[0029] (a) inserting genes selected from NdmA, NdmB, NdmC, NdmD or mixtures thereof in a suitable host,

[0030] (b) fermenting in a suitable media for a suitable time, to obtain a fermentation broth,

[0031] (c) centrifuging and separating the cells, followed by suspending in a suitable buffer at pH 6 - 8 and optimal temperature conditions,

[0032] (d) adding one lot of a substrate xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl; provided that at least one is methyl,

[0033] (e) optionally, checking the consumption of the substrate,

[0034] (f) adding another and further lots of the substrate after every 2 to 8 hours,

[0035] (g) isolating a demethylated product xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl, provided that all of the R1, R2and R3are not methyl at the same time. In another embodiment the gene in step (a) is mixture of NdmA, NdmB and NdmD; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 7-methylxanthine (7 -MX).

[0036] In another embodiment the gene in step (a) is NdmA; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is theobromine or wherein the gene in step (a) is NdmA; the substrate xanthine derivative in step (d) is theophylline and the demethylated product xanthine derivative in step (g) is 3-methylxanthine.

[0037] In another embodiment the gene in step (a) is NdmB; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 1,7- dimethylxanthine, or, wherein the gene in step (a) is NdmB; the substrate xanthine derivative in step (d) is theobromine and the demethylated product xanthine derivative in step (g) is 7-methylxanthine, or, wherein the gene in step (a) is NdmB; the substrate xanthine derivative in step (d) is theophylline and the demethylated product xanthine derivative in step (g) is 1 -methylxanthine.

[0038] In another embodiment the gene in step (a) is mixture of NdmA and NdmB; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 7-methylxanthine, or, wherein the gene in step (a) is mixture of NdmA and NdmB; the substrate xanthine derivative in step (d) is mixture of caffeine and theobromine and the demethylated product xanthine derivative in step (g) is 7-methylxanthine.

[0039] In another embodiment the gene in step (a) is NdmC; the substrate xanthine derivative in step step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 1,3- dimethylxanthine, or, wherein the gene in step (a) is NdmC; the substrate xanthine derivative in step (d) is theobromine and the demethylated product xanthine derivative in step (g) is 3-methylxanthine.

[0040] In all the above demethylation reactions, NdmD is required to catalyze the respective reactions.

[0041] All the above embodiments can alternatively be carried out after extracting the suitable enzymes from the fermentation broth, where in

[0042] (a) inserting genes selected from NdmA, NdmB, NdmC, NdmD or mixtures thereof in a suitable host,

[0043] (b) fermenting in a suitable media for a suitable time, to obtain a fermentation broth,

[0044] (c) centrifuging and separating the cells and carrying out cell lysis,

[0045] (d) adding one lot of a substrate xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl; provided that at least one is methyl,

[0046] (e) optionally, checking the consumption of the substrate,

[0047] (f) adding another and further lots of the substrate after every 2 to 8 hours,

[0048] (g) isolating a demethylated product xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl, provided that all of the R1, R2and R3are not methyl at the same time, wherein the cell pellet or the cell lysate is used directly or immobilized in a suitable matrix in step (d).

[0049] In one embodiment, the specification discloses a demethylated product xanthine derivatives, selected from xanthine, 7-m ethylxanthine (7-MX), theobromine, theophylline, 3- m ethylxanthine, paraxanthine or mixtures thereof, preferably 7-m ethylxanthine (7-MX), is obtained by the fermentation process according to the present disclosure.

[0050] In one embodiment, the specification discloses a demethylated product xanthine derivatives, selected from xanthine, 7-methylxanthine (7-MX), theobromine, theophylline, 3 -methylxanthine, paraxanthine or mixtures thereof, preferably 7-methylxanthine (7-MX), is obtainable by the fermentation process according to the present disclosure.

[0051] BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1: Schematic representation of fermentation process according to Example 1

[0053] Figure 2: Schematic representation of fermentation process according to Example 3

[0054] Figure 3: Schematic representation of fermentation process according to Example 4 DESCRIPTION OF THE INVENTION

[0055] The disclosure generally relates to a fermentation process for the preparation of xanthine or xanthine derivatives such as 7-methylxanthine (7-MX), theobromine, theophylline, 3- methylxanthine.

[0056] The terms ‘7-Methylxanthine’ or ‘7-MX’ relate to same chemical compound, and are used throughout the specification interchangeably, the formula of which is C6H6N4O2 (IUPAC name 7- Methyl-3,7-dihydro-lH-purine-2, 6-dione) as represented by structure below.

[0057] The specification discloses a fermentation process for the preparation of xanthine or xanthine derivatives comprising;

[0058] (a) inserting genes selected from NdmA, NdmB, NdmC, NdmD or mixtures thereof in a suitable host,

[0059] (b) fermenting in a suitable media for a suitable time, to obtain a fermentation broth,

[0060] (c) optionally, centrifuging and separating the cells, followed by suspension in a buffer with pH between 5 to 8 under optimal temperature conditions,

[0061] (d) adding one lot of a substrate xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl; provided that at least one is methyl,

[0062] (e) optionally, checking the consumption of the substrate,

[0063] (f) adding another and further lots of the substrate after every 2 to 8 hours,

[0064] (g) isolating a demethylated product xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl, provided that all of the R1, R2and R3are not methyl at the same time.

[0065] Methylxanthine N-dem ethyl ase (Ndm) possesses broad substrate specificity. In one embodiment, caffeine can be demethylated to theobromine by using NdmA. In another embodiment, theophylline (1,3 -dimethylxanthine) can be demethylated to 3 -methylxanthine by using NdmA. In another embodiment, caffeine can be demethylated to 1,7-m ethylxanthine by using NdmB. In another embodiment, theobromine can be demethylated to 7-methylxanthine by using NdmB. In another embodiment, theophylline can be demethylated to 1 -methylxanthine by using NdmB. In another embodiment, caffeine can be demethylated to 7-methylxanthine by using mixture of NdmA and NdmB. In another embodiment, mixture of caffeine and theobromine can be demethylated to 7-methylxanthine by using mixture of NdmA and NdmB. In another embodiment, caffeine can be demethylated to 1,3 -dimethylxanthine by using NdmC. In another embodiment, theobromine can be demethylated to 3 -methylxanthine by using NdmC. In another embodiment, mixture of caffeine can be demethylated to 7-methylxanthine by using mixture of NdmA and NdmB. In all the above demethylation reactions, NdmD is required to catalyze the respective reactions.

[0066] The substrate xanthine derivative is selected from caffeine, 7-methylxanthine (7 -MX), theobromine, theophylline, 3 -methylxanthine, paraxanthine or mixtures thereof.

[0067] The demethylated product xanthine derivative is selected from xanthine, 7-methylxanthine (7-MX), theobromine, theophylline, 3 -methylxanthine, paraxanthine or mixtures thereof, preferably 7-methylxanthine (7-MX).

[0068] The demethylated product xanthine derivative, selected from xanthine, 7-methylxanthine (7-MX), theophylline, 3 -methylxanthine, paraxanthine or mixtures thereof, preferably 7- methylxanthine (7-MX), is characterized by one or more of the following:

[0069] (i) having an assay by HPLC on dried basis not less than 98% and not more than 102%, preferably at least 99%, more preferably at least 99.5%;

[0070] (ii) having not more than 0.1% of caffeine as an impurity;

[0071] (iii) having not more than 0.1% of theobromine as an impurity;

[0072] (iv) having not more than 0.1% of any unspecified impurities; or

[0073] (v) having not more than 0.5% of total impurities.

[0074] The demethylated product xanthine derivative, selected from xanthine, 7-methylxanthine (7-MX), theobromine, theophylline, 3 -methylxanthine, paraxanthine or mixtures thereof, preferably 7-methylxanthine (7-MX), is characterized by one or more of the following:

[0075] (i) having total aerobic microbial count (TAMC) not more than 103cfu / g, preferably less than 10 cfu / g; (ii) having total combined yeasts / moulds count (TYMC) not more than 102cfu / g, preferably less than 10 cfu / g;

[0076] (iii) having bile-tolerant gram negative bacteria not more than 102cfu / g, preferably less than 10 cfu / g, more preferably absent / g;

[0077] (iv) having Escherichia coli and Staphylococcus aureus absent / g; or

[0078] (v) having Salmonella absent / lOg.

[0079] The demethylated product xanthine derivative, selected from xanthine, 7-methylxanthine (7-MX), theobromine, theophylline, 3 -methylxanthine, paraxanthine or mixtures thereof, preferably 7-methylxanthine (7-MX), is characterized by one or more of the following:

[0080] (i) having particle size between 10 microns to 500 microns, as measured by Malvern 3000 particle size analyser or as per USP 786 method or combination thereof;

[0081] (ii) having loose density of about 0.4 - 0.7 g / ml;

[0082] (iii) having tapped density of about 0.66 - 0.8 g / ml;

[0083] (iv) having loss on drying not more than 0.5%, preferably 0. 12%; or

[0084] (v) having residue on ignition not more than 0.1%, preferably 0.01%.

[0085] The host is selected from bacteria, yeast or pichia, preferably E. coli.

[0086] The addition of each lot of substrate is performed in 2 to 8 hours. Optionally, the addition of each lot of substrate is performed by checking the consumption of the substrate. Each lot of substrate is preferably of about 0.25 molar.

[0087] Isolation of the demethylated product can be performed after addition of preferably at least 5 lots, 10 lots, 20 lots, 40 lots, 90 lots, 120 lots or 240 lots of substrate.

[0088] In the continuous fermentation process as disclosed in the specification, for every 1 litre of fermentation medium, 167 ml of medium is harvested and replenished with additional fermentation broth of step (b). Each harvesting is performed after addition of at least 10 lots of substrate.

[0089] In one embodiment, one harvesting is performed after addition of at least 40 lots of substrate, another harvesting is performed after addition of at least 20 lots of substrate and further harvestings are performed after addition of at least 10 lots of substrate.

[0090] In one embodiment, the present disclosure relates to a demethylated product xanthine derivatives obtained by the fermentation process comprising;

[0091] (a) inserting genes selected from NdmA, NdmB, NdmC, NdmD or mixtures thereof in a suitable host,

[0092] (b) fermenting in a suitable media for a suitable time, to obtain a fermentation broth,

[0093] (c) optionally, centrifuging and separating the cells, followed by suspension in a buffer with pH between 6 to 8 under optimal temperature conditions, (d) adding one lot of a substrate xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl; provided that at least one is methyl,

[0094] (e) optionally, checking the consumption of the substrate,

[0095] (f) adding another and further lots of the substrate after every 2 to 8 hours,

[0096] (g) isolating a demethylated product xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl, provided that all of the R1, R2and R3are not methyl at the same time.

[0097] In another embodiment, the present disclosure relates to a demethylated product xanthine derivatives obtainable by the fermentation process comprising;

[0098] (a) inserting genes selected from NdmA, NdmB, NdmC, NdmD or mixtures thereof in a suitable host,

[0099] (b) fermenting in a suitable media for a suitable time, to obtain a fermentation broth,

[0100] (c) optionally, centrifuging and separating the cells, followed by suspension in a buffer with pH between 6 to 8 under optimal temperature conditions,

[0101] (d) adding one lot of a substrate xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl; provided that at least one is methyl,

[0102] (e) optionally, checking the consumption of the substrate,

[0103] (f) adding another and further lots of the substrate after every 2 to 8 hours,

[0104] (g) isolating a demethylated product xanthine derivative of formula I wherein R1, R2and R3are selected from H or methyl, provided that all of the R1, R2and R3are not methyl at the same time.

[0105] In one embodiment, the demethylated product xanthine derivative is selected from xanthine, 7-m ethylxanthine (7-MX), theobromine, theophylline, 3 -methylxanthine, paraxanthine or mixtures thereof, preferably 7-methylxanthine (7-MX).

[0106] In one embodiment, 7-methylxanthine (7-MX) is characterized by one or more of the following:

[0107] (i) having an assay by HPLC on dried basis not less than 98% and not more than 102%, preferably at least 99%, more preferably at least 99.5%;

[0108] (ii) having not more than 0.1% of caffeine as an impurity;

[0109] (iii) having not more than 0.1% of theobromine as an impurity;

[0110] (iv) having not more than 0.1% of any unspecified impurities; or

[0111] (v) having not more than 0.5% of total impurities.

[0112] In one embodiment, 7-methylxanthine (7-MX) is characterized by one or more of the following:

[0113] (i) having total aerobic microbial count (TAMC) not more than 103cfu / g, preferably less than 10 cfu / g;

[0114] (ii) having total combined yeasts / moulds count (TYMC) not more than 102cfu / g, preferably less than 10 cfu / g;

[0115] (iii) having bile-tolerant gram negative bacteria not more than 102cfu / g, preferably less than 10 cfu / g, more preferably absent / g;

[0116] (iv) having Escherichia coli and Staphylococcus aureus absent / g; or

[0117] (v) having Salmonella absent / lOg.

[0118] In one embodiment, 7-methylxanthine (7-MX) characterized by one or more of the following:

[0119] (i) having particle size between 10 microns to 500 microns, as measure by Malvern 3000 particle size analyser or as per USP 786 method or combination thereof;

[0120] (ii) having loose density of about 0.4 - 0.7 g / ml; or

[0121] (iii) having tapped density of about 0.66 - 0.8 g / ml;

[0122] (iv) having loss on drying not more than 0.5%, preferably 0. 12%; or (v) having residue on ignition not more than 0.1%, preferably 0.01%. In one embodiment, 7-m ethylxanthine (7-MX) is obtained by the process according to the present disclosure.

[0123] In another embodiment, 7-methylxanthine (7-MX) is obtainable by the process according to the present disclosure.

[0124] In one embodiment, 7-methylxanthine (7-MX) as obtained by the process according to the present disclosure is in amorphous form.

[0125] In one embodiment, 7-methylxanthine (7-MX) as obtained by the process according to the present disclosure is in crystalline form.

[0126] In a preferred embodiment, NdmA, Ndmb, NdmC and NdmD genes are isolated from Pseudomonas putida strain CBB5. More preferably, the NdmA gene is of SEQ ID No. 1, NdmB gene is of SEQ ID No. 2, NdmC gene is of SEQ Id No. 3, and NdmD gene is of SEQ ID No. 4.

[0127] The inventors have surprisingly discovered the semi continuous / continuous fermentation processes for the preparation of demethylated products with improved yield and increased efficiency on industrial scale. Advantages of fermentation process as disclosed in the specification over the batch process known in the art are as follows:

[0128] • Higher yield and productivity - prior art process yield per batch is 24% as compared to about 75% as per the invention as disclosed in the specification.

[0129] • Better control of process parameters. The process parameters of resting cell experiments involve separation of cells and reactions at lower temperatures of less than 4 °C. Further the handling of cells ascetically outside the fermenter is critical.

[0130] • No need to clean the fermenter after each product extraction thus saving water, resources & reducing cost.

[0131] • Growth rates are regulated and maintained for extended periods - The conversion of substrate to desired products are being done at the same time, the cells population inside the fermenter are also maintained. This means there is continuous growth of organism and production of the required proteins which enables the conversion to desired products. This is not achieved in a resting cell - batch process whereas in a continuous process, the growth rates are maintained along with the reaction rates for product formation simultaneously.

[0132] • Fermentation broth concentration can be controlled by varying the dilution rate.

[0133] • Growth-limiting nutrients can be maintained at steady-state concentration. The nutrients required for the growth and maintenance of the organism is well maintained in the fermenter compared to a resting cell batch process. In the fermenter, the required nutrients can be provided from time to time compared to a harvested cell / resting cell conversion system • Microorganisms can grow at submaximal rates.

[0134] • Small footprint - small fermenters without the need of large multiple fermenters can be used.

[0135] The fermentation process can be continued as long as there is conversion of substrate to the desired demethylated product. The continuous fermentation process may be terminated if there is no substantial conversion of substrate to the desired demethylated product.

[0136] EXAMPLES

[0137] The following examples illustrate specific embodiments; however, the full scope of the disclosure is not limited to the examples described below.

[0138] HPLC - Related substances method:

[0139] Preparation of Mobile phase A:

[0140] Dissolve 1.36g of Sodium acetate trihydrate in 900 ml of water and mix, add 5 ml of glacial acetic acid and dilute to 1000 ml with water and mix well.

[0141] Preparation of Mobile phase B:

[0142] Prepare a degassed mixture of Mobile phase A and Acetonitrile in the ratio 20:80 % v / v.

[0143] Preparation of diluent:

[0144] Use Water as diluent.

[0145] 5M Ammonia solution:

[0146] Dilute 34 ml of 25% Ammonia solution in 100 ml of water and mix well.

[0147] Elution order:

[0148] Assay (By HPLC) (on dried basis):

[0149] Mobile Phase:

[0150] Dissolve 1.36g of dipotassium hydrogen orthophosphate in 1000 ml of water. To 850ml of this buffer add 150ml of methanol. Then adjust pH to 3.0 with Orthophospharic acid.

[0151] Preparation of 5M ammonia solution:

[0152] Dilute 34 mL of 25 % ammonia solution to 100 mL with water and mix well

[0153] Diluent:

[0154] Use water as Diluent

[0155] Chromatographic Conditions:

[0156] Column : C18, 150mm x 4.6mm, 5p

[0157] Flow rate : 1.0 ml / min

[0158] Detection : UV, 220 nm

[0159] Injection volume : 10 pl

[0160] Run time : 10 minutes

[0161] Particle size:

[0162] The particle size analysis below 150 microns is analyzed by particle size analyzer - Malvern 3000. For particles above 150 microns, sieve test as per USP 786 method is performed.

[0163] Example 1

[0164] Continuous fermentation process for the preparation of 7-MX

[0165] Seed inoculum preparation:

[0166] The genetically modified culture with expressed proteins of ndmA; ndmB; ndmD is inoculated in 50 ml of Luria Bertani (LB) (10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl broth in 250 ml Erlenmeyer flask and run on shaker at 200 RPM, 37 °C for 24 hours. 5 ml of the grown culture is then aseptically transferred to 500ml of LB medium in 2 L Erlenmeyer flask and again run on shaker at 200 RPM, 37 °C for 24 hours.

[0167] Further the 500 ml inoculum generated is inoculated in 75 L fermenter containing 50L LB medium and grown for 24 hours. Temperature was maintained at 37 °C, RPM - 1.2 m / sec.

[0168] Production of 7 MX by continuous fermentation:

[0169] 10,000 L fermenter was charged with 5,000 L of media containing Tryptone - 12g / L; Yeast extract 24g / L; K2HPO4 - 12.54g / L; KH2PO4 - 2.32g / L; Dextrose lOg / L and sterilized at 121 °C using steam and maintained for 30 minutes. The fermenter was then cooled to 37 °C and inoculated with 50 L of seed inoculum generated above.

[0170] Fermentation conditions - 37 °C, Stirring 1.2 m / sec. The optical density reached 17 at the age of 15 hours.

[0171] The temperature was then reduced to 18 °C and IPTG (Isopropyl P- d-1- thiogalactopyranoside) induction was done. 2.4 kg of caffeine in 50 L water pre-sterilized was added. Dextrose was added periodically based on pH increase which indicates demand of dextrose. The conversion of caffeine to 7 MX was checked periodically. The first conversion happened within 3 hours of caffeine addition. Then the second addition of caffeine was done immediately with same volume as first addition, and conversion was continued. Post conversion, addition of caffeine was continued repeatedly for 40 times. After the 40thconversion to 7 MX, 800 L of fermented broth was partially harvested and taken for downstream processing for 7 MX isolation. In the meantime, 800L freshly IPTG induced fermentation broth from another fermenter was added to the main 10 KL fermenter. Then caffeine addition and 7MX conversion was continued for another 20 times repeatedly. After that, another 800 L of broth was partially harvested and replenished with fresh IPTG induced fermentation broth. Another 13 additions of caffeine were carried out for 7MX conversion and again 800 L of broth was partially harvested and replenished with fresh fermentation broth. Caffeine addition was continued till a total of 94 additions, each of 4.8% caffeine solution and then the fermentation was stopped. All the partial harvested broth were simultaneously processed in the downstream for 7MX isolation. Total quantity of caffeine added for 94 additions was 225 kg and the yield of 7 MX from the continuous fermentation was found to be 122 kg.

[0172] Example 2

[0173] Fed batch fermentation for conversion of caffeine to 7-methylxanthine (7-MX)

[0174] Seed inoculum preparation: followed as per Example 1

[0175] Production of 7 Methyl Xanthine (7-MX): 10,000 L fermenter was charged with 5,000 L of media containing Tryptone - 12g / L; Yeast extract 24g / L; K2HPO4 - 2.31g / L; KH2PO4 - 12.54g / L; Dextrose lOg / L and sterilized at 121 °C using steam and maintained for 30 minutes. The fermenter was then cooled to 37 °C and inoculated with 50 L of seed inoculum generated above. Fermentation conditions - 37 °C, RPM 1.2 m / sec. Fermentation was continued till the OD (optical density) reached 15. This happened at 14 hrs post inoculation of the culture. At this stage, temperature was reduced to 18 °C and induced IPTG is added for more protein production (Isopropyl P- d-1 -thiogalactopyranoside). After 2 hours, 4.8% concentration of Caffeine dissolved in water (i.e. 2.416 kg of caffeine in 50 L water pre-sterilized / 0.25 molar concentration) is added to the fermenter aseptically. The fermenter broth sample was periodically checked for conversion of caffeine to 7 methyl xanthine. Once the conversion is completed, the next charge of 4.8% concentration of caffeine in 50L water was added to the broth and again checked for conversion completion. This charging and checking of caffeine are continued periodically till a stage wherein the caffeine conversion to 7 MX stopped. This happened after 44 such periodic additions (44 lots) of caffeine. The total quantity of caffeine charged was 102 kg and the quantity of 7 MX produced was 54 kg. The fermentation broth was taken for downstream purification and product isolation.

[0176] Example-3

[0177] Resting cells for conversion of caffeine to 7-methylxanthine (7-MX)

[0178] The genetically modified culture with expressed proteins of ndmA; ndmB; ndmC is inoculated in 50 ml of Luria Bertani (LB) ( 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl broth in 250 ml Erlenmeyer flask and run on shaker at 200 RPM, 37 °C for 24 hours.

[0179] 30 ml of the above culture was inoculated in 3 L of LB media taken in 5 L fermenter. The fermenter was maintained at 37 °C and 300 RPM stirring. The PH was maintained at 7.0. After about 13 hrs, when the OD reached 10, 7.15 mg of IPTG induction was done and temperature was reduced to 18 °C.

[0180] Fermentation was continued till 24 hrs. Then the fermentation was stopped and the broth was cooled to 4 °C and centrifuged at 6000 RPM. The supernatant was discarded, and the cells were washed with phosphate buffer at pH 7. The cells were then re-suspended in 500 ml phosphate buffer - pH 7.0. 0.25 molar concentration of caffeine was then added to the suspension and was stirred at 30 °C for 4 hours. Then further addition of 0.25 molar concentration caffeine was continued for 10 repeated additions. Each addition of caffeine and conversion of 7MX took about 3-4 hrs. The conversion of caffeine to 7 MX was monitored by HPLC analysis. The reaction was stopped. The purification yielded 75% of caffeine input.

[0181] Example 4: Fermentation media composition and protocol of fermentation up to isolation of cells by centrifugation was followed as per Example 3. Then the cell pellet collected was suspended in 500 ml phosphate buffer at pH 7 and subjected to ultrasound treatment with temperature controlled at less than 5 °C. The cells were checked for lysis microscopically and complete lysis was observed after 2 hours of sonication. The suspension was then centrifuged below 4 °C to separate the cell debris.

[0182] The collected supernatant was then passed on through 300 ml Diaion CR11 resin packed in a glass column. The flow through was circulated at least 5 times in the column to facilitate complete binding of the enzymes on to the resin. Then the resin was washed with pH 5 tris-HCl buffer and then 200ml of 0.25 M caffeine solution with NADH (nicotinamide adenine dinucleotide plus hydrogen) was passed and recirculated through the resin column till complete conversion to 7 MX. Once the conversion was completed, the resin was washed with buffer solution and the next lot of 200 ml of caffeine solution with NADH was recirculated to facilitate conversion to 7 MX. This addition of caffeine solution and NADH was repeated post conversion to 7 MX for 12 times. The converted 7 MX fractions were then pooled together and taken for downstream processing.

[0183] The 7MX product as obtained according to any of the above examples was tested for various criteria and was found to be well within the acceptable limits. Following Table 1 provides the tests conducted and results thereof:

[0184] Comparative Example 1: -

[0185] Resting cells for conversion of caffeine to 7-methylxanthine (7-MX)

[0186] The genetically modified culture with expressed proteins of ndmA; ndmB; ndmC is inoculated in 50 ml of Luria Bertani (LB) ( 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl broth in 250 ml Erlenmeyer flask and run on shaker at 200 RPM, 37 °C for 24 hours.

[0187] 30 ml of the above culture was inoculated in 3 L of LB media taken in 5 L fermenter. The fermenter was maintained at 37 °C and 300 RPM stirring. The PH was maintained at 7.0. After about 13 hrs, when the OD reached 10, 7.15 mg of IPTG induction was done and temperature was reduced to 18 °C.

[0188] Fermentation was continued till 24 hrs. Then the fermentation was stopped and the broth was cooled to 4 °C and centrifuged at 6000 RPM. The supernatant was discarded, and the cells were washed with phosphate buffer at pH 7. The cells were then re-suspended in 500 ml phosphate buffer - pH 7.0. 0.25 molar concentration of caffeine was then added to the suspension and was stirred at 30 °C for 4 hours. The conversion of caffeine to 7 MX was monitored by HPLC analysis. The reaction was stopped. The purification yielded was 30% of caffeine input.

Claims

CLAIMS1. A fermentation process for the preparation of xanthine or xanthine derivatives comprising:(a) inserting genes selected from NdmA, NdmB, NdmC, NdmD or mixtures thereof in a suitable host,(b) fermenting in a suitable media for a suitable time, to obtain a fermentation broth,(c) optionally, centrifuging and separating the cells, followed by suspending in a suitable buffer with pH between 6 to 8 under optimal temperature conditions,(d) adding one lot of a substrate xanthine derivative of formula Iwherein R1, R2and R3are selected from H or methyl; provided that at least one is methyl,(e) optionally, checking the consumption of the substrate,(f) adding another and further lots of the substrate after every 2 to 8 hours,(g) isolating a demethylated product xanthine derivative of formula Iwherein R1, R2and R3are selected from H or methyl, provided that all of the R1, R2and R3are not methyl at the same time.

2. The process according to claim 1, wherein step (c) further comprises cell lysis to obtain cell lysis suspension.

3. The process according to claim 1, wherein step (c) further comprises immobilization of enzyme.

4. The process according to claim 1, wherein the substrate xanthine derivative is selected from caffeine, 7-m ethylxanthine (7-MX), theobromine, theophylline, 3 -methylxanthine, paraxanthine or mixtures thereof.

5. The process according to claim 1, wherein demethylated product xanthine derivative is selected from xanthine, 7-methylxanthine (7-MX), theobromine, theophylline, 3- methylxanthine, paraxanthine or mixtures thereof.

6. The process according to claim 1, wherein the host is selected from bacteria, yeast or pichia.

7. The process according to claim 6, wherein the host is E. coli bacteria B121.

8. The process according to claim 1, wherein the step (b) media is Luria Bertani (LB).

9. The process according to claim 1, wherein each lot of substrates is of about 0.25 molar.

10. The process according to claim 1, wherein isolation of product is performed after addition of at least 5 lots of substrates.

11. The process according to claim 1, wherein isolation of product is performed after addition of at least 20 lots of substrates.

12. The process according to claim 1, wherein isolation of product is performed after addition of at least 40 lots of substrates.

13. The process according to claim 1, wherein isolation of product is performed after addition of at least 90 lots of substrates.

14. The process according to claim 1, wherein isolation of product is performed after addition of at least 120 lots of substrates.

15. The process according to claim 1, wherein isolation of product is performed after addition of at least 240 lots of substrates.

16. The process according to claim 1, wherein for every 1 liter of fermentation medium about 167 ml of fermentation broth is harvested and replenished with additional fermentation broth of step (b).

17. The process according to claim 1, wherein each isolation of product is performed after addition of at least 10 lots of substrates.

18. The process according to claim 1, wherein one isolation of product is performed after addition of at least 40 lots of substrates, another isolation of product is performed after addition of at least 20 lots of substrates and further isolation of product are performed after addition of at least 10 lots of substrates.

19. The process according to claim 1, wherein the gene in step (a) is mixture of NdmA, NdmB and NdmD; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 7-methylxanthine (7-MX).

20. The process according to claim 1, wherein the gene in step (a) is NdmA; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is theobromine or wherein the gene in step (a) is NdmA; the substrate xanthine derivative in step (d) is theophylline and the demethylated product xanthine derivative in step (g) is 3 -methylxanthine.

21. The process according to claim 1, wherein the gene in step (a) is NdmB; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 1,7-dimethylxanthine, or,wherein the gene in step (a) is NdmB; the substrate xanthine derivative in step (d) is theobromine and the demethylated product xanthine derivative in step (g) is 7- methylxanthine, or, wherein the gene in step (a) is NdmB; the substrate xanthine derivative in step (d) is theophylline and the demethylated product xanthine derivative in step (g) is 1- methylxanthine.

22. The process according to claim 1, wherein the gene in step (a) is mixture of NdmA and NdmB; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 7-methylxanthine, or, wherein the gene in step (a) is mixture of NdmA and NdmB; the substrate xanthine derivative in step (d) is mixture of caffeine and theobromine and the demethylated product xanthine derivative in step (g) is 7-methylxanthine.

23. The process according to claim 1, wherein the gene in step (a) is NdmC; the substrate xanthine derivative in step (d) is caffeine and the demethylated product xanthine derivative in step (g) is 1,3 -dimethylxanthine, or, wherein the gene in step (a) is NdmC; the substrate xanthine derivative in step (d) is theobromine and the demethylated product xanthine derivative in step (g) is 3- methylxanthine.

24. A demethylated product xanthine derivative is obtained according to the fermentation process of any of the preceding claims 1-23; wherein, the demethylated product xanthine derivative is 7-methylxanthine (7 -MX).

25. The demethylated product xanthine derivative according to claim 24, wherein 7- methylxanthine (7-MX) is in amorphous form.

26. The demethylated product xanthine derivative according to claim 24, wherein 7- methylxanthine (7-MX) is in crystalline form.

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