Title of Invention	A PROCESS FOR MICROBIAL BIOTRANSFORMATION OF CAFFEINE TO THEOPHYLLINE
Abstract	The present invention is related to " a process for microbial biotransformation of caffeine to theophylline". The main feature of the invention is the production of theophylline by microorganism at room temperature in a simple medium using caffeine as the starting material.Theophylline (C7H8N4O2) 3,7-Dihydro-l,3-dimethyl-lH-purine-2,6-dione; 1,3 dimethyl xanthine having molecular weight 180.17 is an alkaloid naturally present in tea. The importance of theophylline is because of it"s structural similarity with purine base adenine which is an entity of the hereditary system of living organisms.

Title of Invention

A PROCESS FOR MICROBIAL BIOTRANSFORMATION OF CAFFEINE TO THEOPHYLLINE

Abstract

Full Text	The present invention is related to " a process for microbial biotransformation of caffeine to theophylline". The main feature of the invention is the production of theophylline by microorganism at room temperature in a simple medium using caffeine as the starting material. Theophylline (C7H8N4O2) 3,7-Dihydro-1,3-dimethyl-lH-purine-2,6-dione; 1,3 dimethyl xanthine having molecular weight 180.17 is an alkaloid naturally present in tea. The importance of theophylline is because of it's structural similarity with purine base adenine which is an entity of the hereditary system of living organisms. Classical pharmacological studies have revealed that methyl xanthines possess various desirable pharmacological properties. Reference may be made to the work of Beavo,J.A., and Reifsnyder,D.H; Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors; Trends in Pharmacological Science.,1990, 11:150-155, which shows that methyl xanthines have the ability to inhibit cyclic nucleotide phosphodiesterases (PDEs). Reference may be made to the work of Daly, J.W; Adenosine receptors: targets for future drugs. J.Medical chemistry.,1982,25:197-207, which shows the antagonistic receptor mediated actions of adenosine. In general the above mentioned pharmacological activities are reduced in derivatives that lack substituents at position 1 or contain substituents at position 7, as compared with the corresponding 1,3 dialkyl xanthines. Thus the order of potency of the naturally occurring methyl xanthines is theophylline>caffeine>theobromine. Therefore biotransformation of naturally occurring caffeine to more potent molecule viz; theophylline was the main objective of the present invention. Theophylline has been shown to possess therapeutic properties such as diuretic, cardiac stimulant, smooth muscle relaxant and has been successfully used for the treatment of asthma, apnea, diuretic and as a central nervous system stimulator. Theophylline shows bronchodilator effects, arising out of its ability for inhibition of phosphodiesterase (PDEs) which catalyze the breakdown of cyclic AMP and GMP to 5' AMP and 5'GMP respectively. Inhibition of PDEs will lead to an accumulation of cyclic AMP and GMP thereby increasing the signal transduction through these pathways and leading to results in increased levels of cyclic AMP promoting smooth muscle relaxation. Besides its ability to stabilize mast cells other anti-inflammatory and immunomodulatory effects of theophylline have been observed. Adenosine-stimulated release of mediators from mast cells, neutrophil activation, induction of 1L-1ß by 1L-1α, synthesis and release of TNF-α and cytokine release from T-lymphocytes are inhibited by Theophylline. The use of theophylline as drug for asthma, as diuretic and cardiac muscle stimulant is known. Recently the structural resemblance of theophylline to adenine is being made use of to venture into the possibilities of it's use as anti viral, anti cancerous, anti tumourous activity. It has been reported to have anti leukemia property as it inhibits δ-P110 enzyme. Reference may be made to the work of G.Menon, A.D.Ingle, R.P.Gude; Tumor regression of B16F10 Melanoma in vivo by prevention of neovascularisation; Study on Theophylline. Cancer Biotherapy and Radiopharmaceuticals. Vol 17, No. 2, 2002, which shows the possible activity of theophylline on in vivo angiogenesis leading to tumor regression. Reference may be made to I.L.Finar's; Organic Chemistry; Vol-2; Stereochemistry and the chemistry of natural products; Chapter-16, Purines and Nucleic acids, P-809, which refers to the commercial production of theophylline by chemical synthesis (Traube's method) for which the starting materials are dimethyl urea and ethyl cyanoacetate. The drawback of the above method of synthesis is that it uses alkali, nitrous acid, various solvents, hazardous chemicals, is time consuming and not eco- friendly. Reference may be made to the work of Schwimmer. S, Kurtzman,JR, and Heftmann E: Caffeine metabolism by Penicillium roqueforti; Archives of Biochemistry and Biophysics 147, 109-113 (1971) wherein they have identified theophylline as the first product of the caffeine metabolism pathway by fungi. However the disadvantage of this work is the inability to accumulate theophylline in significant level and the work addresses only basic research. Reference may be made to the work of M.Hakil, S.Denis, G.Viniegra-Gonzalez and C.Augur; Degradation and product analysis of caffeine and related dimethylxanthines by filamentous fungi in Enzyme Microbial Technology 22:355-359; 1998, wherein they reported the caffeine metabolism by various filamentous fungi where they were able to utilize caffeine upto 60% but the product accumulation was very low. Reference may be made to the work of Yasuhisa Asano, Toshihiro Komeda and Hideaki Yamada; Microbial production of Theobromine from caffeine; Biosci. Biotech. Biochem.; 57 (8), 1286-1289, 1993, wherein Pseudomonas putida No.352 has been used. However the product accumulated was theobromine, which shows lower pharmacological activity. The main objective of the present invention is to provide a process for microbial biotransformation of caffeine to theophylline, which obviates the drawbacks of the methods described above. Another objective of the present invention is to isolate microorganisms, which possess the capability of biotransforming caffeine to theophylline. Yet another objective of the present invention is to design a suitable medium for the production of theophylline. Another objective of the present invention is to optimize fermentation conditions for effective biotransformation of caffeine to theophylline. The novelty of the present invention is the production of theophylline using a microbial culture grown in a liquid nutrient medium. Accordingly the present invention provides for a process for microbial biotransformation of caffeine to theophylline which comprises the steps of: a) mixing about 1 gram of soil sample collected from coffee plantations followed by making of 10 fold serial dilutions ranging from 10-1 to 10-9; b) inoculating separately 0.1- 0.5 ml of the dilutions, obtained in step (a) on to caffeine agar plates; c) incubating the inoculated plates at temperature in the range of 25 - 35 deg C for a period of about 2-10 days; d) selecting the isolated colonies growing on the above plates followed by further purification in order to obtain pure cultures; e) cultivating the pure cultures obtained in step (d) using 500ml flasks containing caffeine mineral liquid medium maintained on a shaker at an angular speed in the range of 150-200 rpm at a temperature in the range of 25-35 deg C for a period in the range of 70-120 hours in order to obtain liquid cultures; f) drawing samples at specific intervals from the liquid cultures obtained in step (e) followed by analysis of the theophylline formed in order to assess the biotransformation efficiency of the organisms; g) selecting the most efficient biotransforming isolate Penicillium citrinium having MTCC No 5215 for theophylline production; h) inoculating the selected organism Penicillium citrinium having MTCC No 5215 on to potato dextrose agar medium slants containing caffeine at the concentration in the range of 0.1 - 1 g/L followed by incubation of the slants at at temperature in the range of 25-30 deg C for a period of about 72-96 hour in order to obtain spores; i) mixing spores obtained in step (h) with sterile distilled water of quantity in the range of 5-15 ml containing Tween-20 at the concentration of 0.5-5 ml/L in order to obtain spore suspension; j) inoculating the spore suspension obtained in step (i) into 500 ml conical flasks containing mineral medium containing about 0.1 percent caffeine followed by incubation on rotary shaker at an angular speed in the range of 150-200 rpm at a temperature in the range of 25-30 deg C for a period of about 120 hours; k) drawing 1 ml aliquots of samples from the flasks at desired intervals of time followed by centrifugation at 10,000 - 15,000 g for a period in the range of 10-15 minutes in order to obtain supernatant; I) analyzing the supernatant obtained in step (k) for theophylline in High Performance Liquid Chromatography C18 column (HPLC-C18 ) using a mixture of Water and Acetonitrile in the ratio of about 8:2 as the mobile phase with isocratic elution at a flow rate of about 1 ml/min, with an UV-visible Spectrophotometric detector set at 273 nm in order to ascertain microbial biotransformation of caffeine to theophylline. In an embodiment of the present invention, the medium used has the following nutrient composition: Nutrient Concentration (in gram per litre) NaH2PO4 0.1-0.5 KH2PO4 0.5-3.0 MgSO4 0.1-0.5 CaCI2 0.1-0.5 Glucose 5-50 caffeine 0.5-5 In an embodiment of the present invention, the time required for production of theophylline is in the range of 140-240 hours under shaking at an angular speed in the range of 150-200 rpm at a temperature in the range of 25-30 deg C. In an embodiment of the present invention, the biotransformation efficiency of caffeine to theophylline obtained is about 89.7 percent. In an embodiment of the present invention the mineral media employed in the cultivation may include in g/L of NaH2PO4 , 0.1-0.5; KH2PO4, 0.5-3.0; MgSO4, 0.1-0.5; CaCI2,, 0.1-0.5; glucose or sucrose or fructose , 5-50 and caffeine 0.5-5 . In another embodiment of the present invention the growth time may be 140-240 hours under agitation at 150-200 rpm at 25-30°C. The procedure for the microbial production of theophylline from caffeine is given in the following flow sheet: Isolation of microorganism from soil ↓ Purification of isolated microorganism Inoculation of isolated microorganism on slants ↓ Spore suspension Inoculation into shake flask Fermentation (shake flask) ↓ Harvesting of biomass Supernatant for analysis by HPLC and TLC Bacterial isolate, Penicillium citrinium, has been deposited in MTCC (Microbial Type Culture Collection) at Institute of Microbial Technology (IMTECH) Chandigarh, INDIA which is one of there Intenational Depository recognized under Budapest traty and the said culture has the accession number MTCC 5215 The following examples are given by way of illustration of the present invention only and should not be construed to limit the scope of the invention. EXAMPLE-1 One gram of the soil sample collected from coffee plantation and processing units was subjected to ten-fold serial dilution (10-1 to 10-9). 0.1ml of the dilutions was inoculated on to each petri plate and incubated at 25°C for 2 days. Isolated colonies growing on the above plates were selected and purified further to obtain pure cultures. These purified cultures are inoculated on to fresh slants of Potato dextrose agar containing 0.03% caffeine with subsequent sub culturing. The purified isolates were inoculated into 500ml conical flasks containing caffeine mineral media for 70 hours at 25°C on a shaker at 150rpm.Samples were drawn form the above liquid cultures at specific intervals and the theophylline formed was analysed to asses the biotransforming efficiency of the organisms. The most efficient biotransforming organism for theophylline production was selected. Potato dextrose agar slants containing 0.01% caffeine were inoculated with the culture of Penicillium citrinum and incubated at 25°C for 72 hours. A spore suspension of the fully-grown slant was prepared by addition of 5 ml of sterile distilled water containing 0.05% v/v of Tween 20. This spore suspension was inoculated into 100 ml of mineral media in 500 ml conical flasks containing mineral media and caffeine and incubating for a specified period of time at 25 °C at 150 rpm on a rotary shaker at pH of 5.6. Samples (1 ml aliquots) were drawn from the flasks at desired intervals of time and subjected to centrifugation at 10,000 g for 10 minutes and analysed for theophylline by HPLC method as described earlier. The bioconversion of caffeine to theophylline was 53.4%. EXAMPLE-2 One gram of the soil sample collected from coffee plantation and processing units was subjected to ten-fold serial dilution (10-1 to 10-9). 0.3ml of the dilutions was inoculated on to each petri plate and incubated at 28°C for 4 days. Isolated colonies growing on the above plates were selected and purified further to obtain pure cultures. These purified cultures are inoculated on to fresh slants of Potato dextrose agar containing 0.03% caffeine with subsequent sub culturing. The purified isolates were inoculated into 500ml conical flasks containing caffeine mineral media for 96 hours at 28°C on a shaker at 200 rpm. Samples were drawn form the above liquid cultures at specific intervals and the theophylline formed was analysed to assess the biotransforming efficiency of the organisms. The most efficient biotransforming organism for theophylline production was selected. Potato dextrose agar slants containing 0.05% caffeine were inoculated with the culture of Penicillium citrinum and incubated at 28°C for 72 hours. A spore suspension of the fully-grown slant was prepared by addition of 5 ml of sterile distilled water containing 0.05% v/v of Tween 20. This spore suspension was inoculated into 100 ml of mineral media in 500 ml conical flasks containing mineral media and caffeine for a specified period of time at 28 °C at 200 rpm on a rotary shaker a pH of 5.6. Samples (1 ml aliquots) were drawn from the flasks at desired intervals of time and subjected to centrifugation at 15,000 g for 15 minutes and analysed for theophylline by HPLC method as described earlier. The bioconversion of caffeine to theophylline was 89.7%. EXAMPLE-3 One gram of the soil sample collected from coffee plantation and processing units was subjected to ten-fold serial dilution (10-1 to 10-9). 0.5ml of the dilutions was inoculated on to each petri plate and incubated at 35°C for 10 days. Isolated colonies growing on the above plates were selected and purified further to obtain pure cultures. These purified cultures are inoculated on to fresh slants of Potato dextrose agar containing 0.03% caffeine with subsequent sub culturing. The purified isolates were inoculated into 500ml conical flasks containing caffeine mineral media for 120 hours at 35°C on a shaker at 200 rpm. Samples were drawn form the above liquid cultures at specific intervals and the theophylline formed was analysed to assess the biotransforming efficiency of the organisms. The most efficient biotransforming organism for theophylline production was selected. Potato dextrose agar slants containing 0.1% caffeine were inoculated with the culture of Penicillium citrinum and incubated at 30°C for 96 hours. A spore suspension of the fully-grown slant was prepared by addition of 15 ml of sterile distilled water containing 0.5% v/v of Tween 20. This spore suspension was inoculated into 100 ml of mineral media in 500 ml conical flasks containing mineral media and caffeine for a specified period of time at 35 °C at 200 rpm on a rotary shaker a pH of 5.6. Samples (1 ml aliquots) were drawn from the flasks at desired intervals of time and subjected to centrifugation at 15,000 g for 15 minutes and analysed for theophylline by HPLC method as described earlier. The bioconversion of caffeine to theophylline was 79.5%. EXAMPLE-4 Biotransformation of Penicillium citrinum having culture No. 5215 .was carried out as in example 1, wherein the nutrients in the mineral media included in g/L of NaH2PO4 , 0.1; KH2PO4, 0.5; MgSO4, 0.1; CaCl2,, 0.1; glucose, 5 and caffeine 0.5. The biotransformation efficiency was 80.3%. EXAMPLE-5 Biotransformation of Penicillium citrinum having culture No. 5215 was carried out as in example 1, wherein the nutrients in the mineral media included in g/L of NaH2PO4 , 0.12; KH2PO4, 01.3; MgSO4, 0.3; CaCI2, 0.3; glucose, 20 and caffeine 1.0. The biotransformation efficiency was 89.7%. EXAMPLE-6 Biotransformation of Penicillium citrinum having MTCC culture No. 5215 .was carried out as in example 1, wherein the nutrients in the mineral media included in g/L of NaH2PO4 , 0.5; KH2PO4, 3.0; MgSO4, 0.5; CaCI2, 0.5; glucose,50 and caffeine 5.0. The biotransformation efficiency was 52.7% Example-7 Biotransformation of caffeine to theophylline was carried out as in example 2 and 5, with the time of fermentation for theophylline production was 140 hours under agitation at 150 rpm at 25°C. The biotransformation efficiency was 62%. Example-8 Biotransformation of caffeine to theophylline was carried out as in example 3 and 6, with the time of fermentation for theophylline production was 196 hours under agitation at 200 rpm at 28°C. The biotransformation efficiency was 89.7%. Example-9 Biotransformation of caffeine to theophylline was carried out as in example 3 and 6, with the time of fermentation for theophylline production was 240 hours under agitation at 200 rpm at 35°C. The biotransformation efficiency was reduced to 73%. The main advantages of the present invention are: 1. It employs simple and inexpensive media for the cultivation of a culture of Penicillium citrinium having culture No 5215 capable of biotransforming caffeine to theophylline. 2. It avoids the use of hazardous organic solvents and other toxic chemicals and hence is environmental friendly. We claim: 1. A biologically pure isolated fungal strain of Penicillium citrinium having accession number MTCC 5215, wherein the said isolate is useful for microbial biotransformation of caffeine to theophylline. 2. A process for microbial biotransformation of caffeine to theophylline using Penicillium citrinium MTCC 5215 as claimed in claim 1, wherein the steps comprising: a) homogenizing 1 gram of soil sample collected from coffee plantations followed by preparing 10 fold serial dilutions ranging from 10"1 to 10"9; b) separately inoculating 0.1 - 0.5 ml of the dilutions as obtained in step (a) on to caffeine agar plates; c) incubating the inoculated plates of step (b) at temperature in the range of 25 -35 degree C for a period of 2-10 days and selecting the isolated colonies followed by further purification to obtain pure cultures; d) inoculating the pure cultures obtained in step (c) individually in 500ml caffeine mineral liquid medium maintained on a shaker at an angular speed in the range of 150-200 rpm at a temperature in the range of 25-35 deg C for a period of 70-120 hours to obtain liquid cultures; e) withdrawing samples from the liquid cultures obtained in step (e) at specific intervals followed by analysis of the theophylline formed to assess the biotransformation efficiency of the cultures; f) selecting the most efficient biotransforming isolate identified as Penicillium citrinium having MTCC No 5215 for theophylline production; g) inoculating the selected organism Penicillium citrinium having MTCC No 5215 of step (f) on to potato dextrose agar medium slants containing caffeine at the concentration in the range of 0.1 -1 g/L followed by incubation at temperature in the range of 25-30 degree C for a period of 72-96 hours to obtain spores; h) mixing the spores as obtained in step (g) with sterile distilled containing 0.5-5 ml/L Tween-20 to obtain spore suspension; i) inoculating the spore suspension as obtained in step (h) into 500 ml mineral medium containing 0.1 percent caffeine followed by incubation on rotary shaker at 150-200 rpm at a temperature in the range of 25-30 deg C for a period of 120-240 hours; j) drawing 1 ml aliquot of sample from the incubated spore suspension of step (i) at desired intervals of time followed by centrifugation at 10,000 - 15,000 g for 10-15 minutes to obtain supernatant; k) analyzing the supernatant obtained in step (j) for theophylline in High Performance Liquid Chromatography C18 column (HPLC-C18 ) using a mixture of Water and Acetonitrile in the ratio of 8:2 as the mobile phase with isocratic elution at a flow rate of 1 ml/min, with an UV-visible Spectrophotometric detector set at 273 nm to ascertain the microbial biotransformation of caffeine to theophylline. 3. A process as claimed in claim 2, wherein the medium used has the following nutrient composition: Nutrient Concentration (gram per litre) NaH2P04 0.1-0.5 KH2P04 0.5-3.0 MgS04 0.1-0.5 CaCI2 0.1-0.5 Glucose 5-50 Caffeine 0.5-5 4. A process as claimed in claim 2, wherein the biotransformation efficiency of caffeine to theophylline obtained is upto 89.7 percent. 5. A process for microbial biotransformation of caffeine to theophylline substantially as herein described in the description and exemplified in the acompanying examples.

Full Text

The present invention is related to " a process for microbial biotransformation of caffeine to theophylline". The main feature of the invention is the production of theophylline by microorganism at room temperature in a simple medium using caffeine as the starting material.
Theophylline (C7H8N4O2) 3,7-Dihydro-1,3-dimethyl-lH-purine-2,6-dione; 1,3 dimethyl xanthine having molecular weight 180.17 is an alkaloid naturally present in tea.
The importance of theophylline is because of it's structural similarity with purine base adenine which is an entity of the hereditary system of living organisms. Classical pharmacological studies have revealed that methyl xanthines possess various desirable pharmacological properties. Reference may be made to the work of Beavo,J.A., and Reifsnyder,D.H; Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors; Trends in Pharmacological Science.,1990, 11:150-155, which shows that methyl xanthines have the ability to inhibit cyclic nucleotide phosphodiesterases (PDEs). Reference may be made to the work of Daly, J.W; Adenosine receptors: targets for future drugs. J.Medical chemistry.,1982,25:197-207, which shows the antagonistic receptor mediated actions of adenosine. In general the above mentioned pharmacological activities are reduced in derivatives that lack substituents at position 1 or contain substituents at position 7, as compared with the corresponding 1,3 dialkyl xanthines. Thus the order of potency of the naturally occurring methyl xanthines is theophylline>caffeine>theobromine. Therefore biotransformation of naturally occurring caffeine to more potent molecule viz; theophylline was the main objective of the present invention.
Theophylline has been shown to possess therapeutic properties such as diuretic, cardiac stimulant, smooth muscle relaxant and has been successfully used for the treatment of asthma, apnea, diuretic and as a central nervous system stimulator. Theophylline shows bronchodilator effects, arising out of its ability for inhibition of phosphodiesterase (PDEs) which catalyze the breakdown of cyclic AMP and GMP to 5' AMP and 5'GMP respectively. Inhibition of PDEs will lead to an accumulation of cyclic AMP and GMP thereby increasing the signal transduction through these pathways and leading to results in increased levels of cyclic AMP promoting smooth muscle relaxation. Besides its ability to stabilize mast cells other anti-inflammatory and immunomodulatory effects of theophylline have been observed. Adenosine-stimulated release of mediators from mast cells, neutrophil activation, induction of 1L-1ß by 1L-1α, synthesis and release of TNF-α and cytokine release from T-lymphocytes are inhibited by Theophylline.
The use of theophylline as drug for asthma, as diuretic and cardiac muscle stimulant is known. Recently the structural resemblance of theophylline to adenine is being made use of to venture into the possibilities of it's use as anti viral, anti cancerous, anti tumourous activity. It has been reported to have anti leukemia property as it inhibits δ-P110 enzyme. Reference may be made to the work of G.Menon, A.D.Ingle, R.P.Gude; Tumor regression of B16F10 Melanoma in vivo by prevention of neovascularisation; Study on Theophylline. Cancer Biotherapy and Radiopharmaceuticals. Vol 17, No. 2, 2002, which shows the possible activity of theophylline on in vivo angiogenesis leading to tumor regression.
Reference may be made to I.L.Finar's; Organic Chemistry; Vol-2; Stereochemistry and the chemistry of natural products; Chapter-16, Purines and Nucleic acids, P-809, which refers to the commercial production of theophylline by chemical synthesis (Traube's method) for which the starting materials are dimethyl urea and ethyl cyanoacetate. The drawback of the above method of synthesis is that it uses alkali, nitrous acid, various solvents, hazardous chemicals, is time consuming and not eco- friendly.
Reference may be made to the work of Schwimmer. S, Kurtzman,JR, and Heftmann E: Caffeine metabolism by Penicillium roqueforti; Archives of Biochemistry and Biophysics 147, 109-113 (1971) wherein they have identified theophylline as the first product of the caffeine metabolism pathway by fungi. However the disadvantage of this work is the inability to accumulate theophylline in significant level and the work addresses only basic research.
Reference may be made to the work of M.Hakil, S.Denis, G.Viniegra-Gonzalez and C.Augur; Degradation and product analysis of caffeine and related dimethylxanthines by filamentous fungi in Enzyme Microbial Technology 22:355-359; 1998, wherein they reported the caffeine metabolism by various filamentous fungi where they were able to utilize caffeine upto 60% but the product accumulation was very low.
Reference may be made to the work of Yasuhisa Asano, Toshihiro Komeda and Hideaki Yamada; Microbial production of Theobromine from caffeine; Biosci. Biotech. Biochem.; 57 (8), 1286-1289, 1993, wherein Pseudomonas putida No.352 has been used. However the product accumulated was theobromine, which shows lower pharmacological activity.
The main objective of the present invention is to provide a process for microbial biotransformation of caffeine to theophylline, which obviates the drawbacks of the methods described above.
Another objective of the present invention is to isolate microorganisms, which possess the capability of biotransforming caffeine to theophylline.
Yet another objective of the present invention is to design a suitable medium for the production of theophylline.
Another objective of the present invention is to optimize fermentation conditions for effective biotransformation of caffeine to theophylline.
The novelty of the present invention is the production of theophylline using a microbial culture grown in a liquid nutrient medium.
Accordingly the present invention provides for a process for microbial biotransformation of caffeine to theophylline which comprises the steps of:
a) mixing about 1 gram of soil sample collected from coffee plantations followed by making of 10 fold serial dilutions ranging from 10-1 to 10-9;
b) inoculating separately 0.1- 0.5 ml of the dilutions, obtained in step (a) on to caffeine agar plates;
c) incubating the inoculated plates at temperature in the range of 25 - 35 deg C for a period of about 2-10 days;
d) selecting the isolated colonies growing on the above plates followed by further purification in order to obtain pure cultures;
e) cultivating the pure cultures obtained in step (d) using 500ml flasks containing caffeine mineral liquid medium maintained on a shaker at an angular speed in the range of 150-200 rpm at a temperature in the range of 25-35 deg C for a period in the range of 70-120 hours in order to obtain liquid cultures;
f) drawing samples at specific intervals from the liquid cultures
obtained in step (e) followed by analysis of the theophylline
formed in order to assess the biotransformation efficiency of the
organisms;
g) selecting the most efficient biotransforming isolate Penicillium
citrinium having MTCC No 5215 for theophylline production;
h) inoculating the selected organism Penicillium citrinium having MTCC No 5215 on to potato dextrose agar medium slants containing caffeine at the concentration in the range of 0.1 - 1 g/L followed by incubation of the slants at at temperature in the range of 25-30 deg C for a period of about 72-96 hour in order to obtain spores;
i) mixing spores obtained in step (h) with sterile distilled water of quantity in the range of 5-15 ml containing Tween-20 at the concentration of 0.5-5 ml/L in order to obtain spore suspension;
j) inoculating the spore suspension obtained in step (i) into 500 ml conical flasks containing mineral medium containing about 0.1 percent caffeine followed by incubation on rotary shaker at an angular speed in the range of 150-200 rpm at a temperature in the range of 25-30 deg C for a period of about 120 hours;
k) drawing 1 ml aliquots of samples from the flasks at desired intervals of time followed by centrifugation at 10,000 - 15,000 g for a period in the range of 10-15 minutes in order to obtain supernatant;
I) analyzing the supernatant obtained in step (k) for theophylline in High Performance Liquid Chromatography C18 column (HPLC-C18 ) using a mixture of Water and Acetonitrile in the ratio of about 8:2 as the mobile phase with isocratic elution at a flow rate of about 1 ml/min, with an UV-visible Spectrophotometric detector set at 273 nm in order to ascertain microbial biotransformation of caffeine to theophylline.
In an embodiment of the present invention, the medium used has the following nutrient composition:
Nutrient Concentration
(in gram per litre)
NaH2PO4 0.1-0.5
KH2PO4 0.5-3.0
MgSO4 0.1-0.5
CaCI2 0.1-0.5
Glucose 5-50
caffeine 0.5-5
In an embodiment of the present invention, the time required for production of theophylline is in the range of 140-240 hours under shaking at an angular speed in the range of 150-200 rpm at a temperature in the range of 25-30 deg C.
In an embodiment of the present invention, the biotransformation efficiency of caffeine to theophylline obtained is about 89.7 percent.
In an embodiment of the present invention the mineral media employed in the cultivation may include in g/L of NaH2PO4 , 0.1-0.5; KH2PO4, 0.5-3.0; MgSO4, 0.1-0.5; CaCI2,, 0.1-0.5; glucose or sucrose or fructose , 5-50 and caffeine 0.5-5 .
In another embodiment of the present invention the growth time may be 140-240 hours under agitation at 150-200 rpm at 25-30°C.
The procedure for the microbial production of theophylline from caffeine is given in the following flow sheet:
Isolation of microorganism from soil
↓
Purification of isolated microorganism
Inoculation of isolated microorganism on slants
↓
Spore suspension Inoculation into shake flask Fermentation (shake flask)
↓
Harvesting of biomass Supernatant for analysis by HPLC and TLC
Bacterial isolate, Penicillium citrinium, has been deposited in MTCC (Microbial Type Culture Collection) at Institute of Microbial Technology (IMTECH) Chandigarh, INDIA which is one of there Intenational Depository recognized under Budapest traty and the said culture has the accession number MTCC 5215
The following examples are given by way of illustration of the present invention only and should not be construed to limit the scope of the invention.
EXAMPLE-1
One gram of the soil sample collected from coffee plantation and processing units was subjected to ten-fold serial dilution (10-1 to 10-9).
0.1ml of the dilutions was inoculated on to each petri plate and incubated at 25°C for 2 days. Isolated colonies growing on the above plates were selected and purified further to obtain pure cultures. These purified cultures are inoculated on to fresh slants of Potato dextrose agar containing 0.03% caffeine with subsequent sub culturing. The purified isolates were inoculated into 500ml conical flasks containing caffeine mineral media for 70 hours at 25°C on a shaker at 150rpm.Samples were drawn form the above liquid cultures at specific intervals and the theophylline formed was analysed to asses the biotransforming efficiency of the organisms. The most efficient biotransforming organism for theophylline production was selected. Potato dextrose agar slants containing 0.01% caffeine were inoculated with the culture of Penicillium citrinum and incubated at 25°C for 72 hours. A spore suspension of the fully-grown slant was prepared by addition of 5 ml of sterile distilled water containing 0.05% v/v of Tween 20. This spore suspension was inoculated into 100 ml of mineral media in 500 ml conical flasks containing mineral media and caffeine and incubating for a specified period of time at 25 °C at 150 rpm on a rotary shaker at pH of 5.6. Samples (1 ml aliquots) were drawn from the flasks at desired intervals of time and subjected to centrifugation at 10,000 g for 10 minutes and analysed for
theophylline by HPLC method as described earlier. The bioconversion of caffeine to theophylline was 53.4%.
EXAMPLE-2
One gram of the soil sample collected from coffee plantation and processing units was subjected to ten-fold serial dilution (10-1 to 10-9). 0.3ml of the dilutions was inoculated on to each petri plate and incubated at 28°C for 4 days. Isolated colonies growing on the above plates were selected and purified further to obtain pure cultures. These purified cultures are inoculated on to fresh slants of Potato dextrose agar containing 0.03% caffeine with subsequent sub culturing. The purified isolates were inoculated into 500ml conical flasks containing caffeine mineral media for 96 hours at 28°C on a shaker at 200 rpm. Samples were drawn form the above liquid cultures at specific intervals and the theophylline formed was analysed to assess the biotransforming efficiency of the organisms. The most efficient biotransforming organism for theophylline production was selected. Potato dextrose agar slants containing 0.05% caffeine were inoculated with the culture of Penicillium citrinum and incubated at 28°C for 72 hours. A spore suspension of the fully-grown slant was prepared by addition of 5 ml of sterile distilled water containing 0.05% v/v of Tween 20. This spore suspension was inoculated into 100 ml of mineral media in 500 ml conical flasks containing mineral media and
caffeine for a specified period of time at 28 °C at 200 rpm on a rotary shaker a pH of 5.6. Samples (1 ml aliquots) were drawn from the flasks at desired intervals of time and subjected to centrifugation at 15,000 g for 15 minutes and analysed for theophylline by HPLC method as described earlier. The bioconversion of caffeine to theophylline was 89.7%.
EXAMPLE-3
One gram of the soil sample collected from coffee plantation and processing units was subjected to ten-fold serial dilution (10-1 to 10-9). 0.5ml of the dilutions was inoculated on to each petri plate and incubated at 35°C for 10 days. Isolated colonies growing on the above plates were selected and purified further to obtain pure cultures. These purified cultures are inoculated on to fresh slants of Potato dextrose agar containing 0.03% caffeine with subsequent sub culturing. The purified isolates were inoculated into 500ml conical flasks containing caffeine mineral media for 120 hours at 35°C on a shaker at 200 rpm. Samples were drawn form the above liquid cultures at specific intervals and the theophylline formed was analysed to assess the biotransforming efficiency of the organisms. The most efficient biotransforming organism for theophylline production was selected.
Potato dextrose agar slants containing 0.1% caffeine were inoculated with the culture of Penicillium citrinum and incubated at 30°C for 96 hours. A spore suspension of the fully-grown slant was prepared by addition of 15 ml of sterile distilled water containing 0.5% v/v of Tween 20. This spore suspension was inoculated into 100 ml of mineral media in 500 ml conical flasks containing mineral media and caffeine for a specified period of time at 35 °C at 200 rpm on a rotary shaker a pH of 5.6. Samples (1 ml aliquots) were drawn from the flasks at desired intervals of time and subjected to centrifugation at 15,000 g for 15 minutes and analysed for theophylline by HPLC method as described earlier. The bioconversion of caffeine to theophylline was 79.5%.
EXAMPLE-4
Biotransformation of Penicillium citrinum having culture No. 5215 .was carried out as in example 1, wherein the nutrients in the mineral media included in g/L of NaH2PO4 , 0.1; KH2PO4, 0.5; MgSO4, 0.1; CaCl2,, 0.1; glucose, 5 and caffeine 0.5. The biotransformation efficiency was 80.3%.
EXAMPLE-5
Biotransformation of Penicillium citrinum having culture No. 5215 was carried out as in example 1, wherein the nutrients in the mineral
media included in g/L of NaH2PO4 , 0.12; KH2PO4, 01.3; MgSO4, 0.3; CaCI2, 0.3; glucose, 20 and caffeine 1.0. The biotransformation efficiency was 89.7%.
EXAMPLE-6
Biotransformation of Penicillium citrinum having MTCC culture No. 5215 .was carried out as in example 1, wherein the nutrients in the mineral media included in g/L of NaH2PO4 , 0.5; KH2PO4, 3.0; MgSO4, 0.5; CaCI2, 0.5; glucose,50 and caffeine 5.0. The biotransformation efficiency was 52.7%
Example-7
Biotransformation of caffeine to theophylline was carried out as in example 2 and 5, with the time of fermentation for theophylline production was 140 hours under agitation at 150 rpm at 25°C. The biotransformation efficiency was 62%.
Example-8
Biotransformation of caffeine to theophylline was carried out as in example 3 and 6, with the time of fermentation for theophylline production was 196 hours under agitation at 200 rpm at 28°C. The biotransformation efficiency was 89.7%.
Example-9
Biotransformation of caffeine to theophylline was carried out as in example 3 and 6, with the time of fermentation for theophylline production was 240 hours under agitation at 200 rpm at 35°C. The biotransformation efficiency was reduced to 73%.
The main advantages of the present invention are:
1. It employs simple and inexpensive media for the cultivation of a culture of Penicillium citrinium having culture No 5215 capable of biotransforming caffeine to theophylline.
2. It avoids the use of hazardous organic solvents and other toxic chemicals and hence is environmental friendly.

We claim:
1. A biologically pure isolated fungal strain of Penicillium citrinium having accession number MTCC 5215, wherein the said isolate is useful for microbial biotransformation of caffeine to theophylline.
2. A process for microbial biotransformation of caffeine to theophylline using Penicillium citrinium MTCC 5215 as claimed in claim 1, wherein the steps comprising:

a) homogenizing 1 gram of soil sample collected from coffee plantations followed by preparing 10 fold serial dilutions ranging from 10"1 to 10"9;
b) separately inoculating 0.1 - 0.5 ml of the dilutions as obtained in step (a) on to caffeine agar plates;
c) incubating the inoculated plates of step (b) at temperature in the range of 25 -35 degree C for a period of 2-10 days and selecting the isolated colonies followed by further purification to obtain pure cultures;
d) inoculating the pure cultures obtained in step (c) individually in 500ml caffeine mineral liquid medium maintained on a shaker at an angular speed in the range of 150-200 rpm at a temperature in the range of 25-35 deg C for a period of 70-120 hours to obtain liquid cultures;
e) withdrawing samples from the liquid cultures obtained in step (e) at specific intervals followed by analysis of the theophylline formed to assess the biotransformation efficiency of the cultures;
f) selecting the most efficient biotransforming isolate identified as Penicillium citrinium having MTCC No 5215 for theophylline production;
g) inoculating the selected organism Penicillium citrinium having MTCC No 5215 of step (f) on to potato dextrose agar medium slants containing caffeine at the concentration in the range of 0.1 -1 g/L followed by incubation at temperature in the range of 25-30 degree C for a period of 72-96 hours to obtain spores;
h) mixing the spores as obtained in step (g) with sterile distilled containing 0.5-5 ml/L Tween-20 to obtain spore suspension;
i) inoculating the spore suspension as obtained in step (h) into 500 ml mineral medium containing 0.1 percent caffeine followed by incubation on rotary shaker at 150-200 rpm at a temperature in the range of 25-30 deg C for a period of 120-240 hours;
j) drawing 1 ml aliquot of sample from the incubated spore suspension of step (i) at desired intervals of time followed by centrifugation at 10,000 - 15,000 g for 10-15 minutes to obtain supernatant;
k) analyzing the supernatant obtained in step (j) for theophylline in High Performance Liquid Chromatography C18 column (HPLC-C18 ) using a mixture of Water and Acetonitrile in the ratio of 8:2 as the mobile phase with isocratic elution at a flow rate of 1 ml/min, with an UV-visible Spectrophotometric detector set at 273 nm to ascertain the microbial biotransformation of caffeine to theophylline.
3. A process as claimed in claim 2, wherein the medium used has
the following nutrient composition:
Nutrient Concentration (gram per litre)
NaH2P04 0.1-0.5
KH2P04 0.5-3.0
MgS04 0.1-0.5
CaCI2 0.1-0.5
Glucose 5-50
Caffeine 0.5-5
4. A process as claimed in claim 2, wherein the biotransformation efficiency of caffeine
to theophylline obtained is upto 89.7 percent.
5. A process for microbial biotransformation of caffeine to theophylline substantially as herein described in the description and exemplified in the acompanying examples.

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Patent Number

250721

Indian Patent Application Number

329/DEL/2006

PG Journal Number

04/2012

Publication Date

27-Jan-2012

Grant Date

23-Jan-2012

Date of Filing

03-Feb-2006

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

ANUSANDHAN BHAWAN,RAFI MARG, NEW DELHI-110 001

Inventors:

#	Inventor's Name	Inventor's Address
1	SANJUKTA PATRA	FERMENTATION TECHNOLOGY AND BIOENGINEERING DEPT. CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE MYSORE KARNATAKA-570020
2	MUNNA SINGH THAKUR	FERMENTATION TECHNOLOGY AND BIOENGINEERING DEPT. CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE MYSORE KARNATAKA-570020
3	NAIKANKATTE GANESH KARANTH	FERMENTATION TECHNOLOGY AND BIOENGINEERING DEPT. CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE MYSORE KARNATAKA-570020

PCT International Classification Number

C12R 1/80

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA