FIELD OF THE INVENTION The instant invention relates to a novel process for the manufacture of Mycophenolic acid by solid state fermentation of Penicillium brevicompactum using fed-batch production technique.

DESCRIPTION OF PRIOR ART Mycophenolic acid (MPA) as a compound with several useful biological properties such as antiviral and antitumor activities is described in non-patent literature (K. Ando et. al., J. Antibiot, 21, 649-652,1968 and R. H. Williams et al., J. Antibiot, 21, 463-464, 1968).

Antimicrobial activity of MPA has also been reported in literature (K.

Gilliver, Ann. Bot. (London) 10, 271-282, 1946).

MPA was initially isolated from a culture of a fungus belonging to the genus Penicillium and it is known that MPA is produced by many species of the genus Penicillium. For example, P. brevi-compactum, P. stroniferum, P scabrum, P nagemi, P Szaferiand P patus-mai, (Biochem. J. 27, 654, 1933).

GB 11507099 describes a process for the production of mycophenolic acid and salts thereof which comprises the submerged aerobic cultivation of a mycophenolic acid-producing species of Penicillium in a nutrient medium providing not less than 0. 001% w/v of magnesium at the beginning of the process, such that the magnesium source is present substantially for the duration of the process to obtain good yields of mycophenolic acid.

US 4, 452, 891 claims a method for producing mycophenolic acid by fermentation, which comprises, (i) aerobically culturing in a culture medium under conditions suitable for the accumulation of said mycophenolic acid, a strain of Penicillium identified as Penicillium brevicompactum FERM BP-53, Penicillium brevicompactum FERM-BP- 54, or Penicillium brevicompactum BP-55 which is capable of producing mycophenolic acid ; (ii) recovering the mycophenolic acid which accumulates in the culture medium.

Optimization of mycophenolic acid production in solid-state fermentation using response surface methodology has been discussed by Sadhukhan etal (J. Ind. Microbiol. Biotechnol. ; 1999, 22, 1, 33-38).

WO 01/64931 discloses a method of manufacturing MPA by solid state fermentation of Penicillium brevicompactum in a contained bioreactor under optimal fermentation conditions.

Solid state fed-batch methods used till now for cellulase and gibberalic acid production involved atomizing ammonium sulphate for feeding (JwChem Technol. Biotechnol. ; (1997) 69, 4, 429-32), shot addition of solid corn starch in a 50 L continuously rotating reactor (Process-Biochem.; (1997) 32, 2, 141-45), shot addition of only carbon source at very small scale (Biotechnol. Lett. ; (1987) 9, 3, 179- 82) Methods for the production of MPA by submerged as well as solid state fermentation route have been reported in literature. However, the need to improve yields for better commercial viability exists.

Producing biological compounds by fermentation, especially fed batch fermentation, in submerged fermentation process is an expensive procedure, as the demand for purified water and clean steam (for sterilization) can be high, the process is generally labor intensive, requiring skilled workers at all levels of production, and the cost of high grade nutrients and other ingredients necessary for culture media can be high.

A need to provide a simple fed batch fermentation system that minimizes the cost for production of the compound of interest by providing required nutrients in proper feed rate, with the right component constitution required during the process and in a cost effective manner. To Maximize yield of the compound of interest and

avoiding problems that may occur in media preparation such as precipitation of certain ingredients or the formation of chemical intermediates during sterilization was felt.

Accordingly, it is an objective of the present invention to provide a fermentation system/process that reduces the cost of production of MPA, by an innovative process of fed-batch fermentation in solid state matrix.

DESCRIPTION Definitions "Solid state fermentation"or"solid state cultivation" : The term"solid state fermentation"or"solid state cultivation", sometimes referred to as"semi-solid state fermentation"as used herein, means the process of fermenting microorganisms on a solid medium that provides anchorage points for the microorganisms in the absence of any freely flowing substance. The amount of water in the solid medium can be any amount of water. For example, the solid medium could be almost dry, or it could be slushy. A person skilled in the art knows that the terms"solid state fermentation"and"semi-solid state fermentation"are interchangeable.

"Fed-batch fermentation"or"fed-batch technique" : The term fed-batch fermentation as used herein, means a fermentation process carried out where substrate or nutrients are added in small

increments as the fermentation progresses. The substrate or nutrient is added in small increment that would encourage the production of secondary metabolites because some secondary metabolite production is inhibited by high concentrations of substrate or substrates, so this method would encourage the production of such metabolites. Supplement of nutrients at a time when the initially fed nutrient are consumed by the microorganisms or culture also help in providing more energy to the microorganism which in turn increases the overall production of the secondary metabolites. However, if the culture is capable of metabolizing nutrients even at higher concentrations, then continuous fed-batch simplifies to semi- continuous fed-batch where the size of increment increase and the frequency of addition get reduced.

"Bioreactor" : The term"bioreactor"as used herein, means a device capable of holding fermentation media inoculated with microorganism and carrying out the process of solid state fermentation in a contained manner. A bioreactor can be used to grow any microorganism capable of growing under specified conditions in a contained environment. Some examples of microorganisms capable of growing in a bioreactor are fungi, yeast and bacteria. Particularly preferred microorganisms are fungi. Fungi that can be used in the present invention include septate as well as aseptate fungi producing either extracellular or intracellular metabolites.

Fed batch fermentation systems are generally defined as batch culture systems wherein fresh nutrients and/or other additives (such as precursors to products) are added but no medium is withdrawn.

The current invention involves a process for the manufacture of mycophenolic acid by solid substrate fermentation involving nutrient feeding for fed batch fermentation to enhance productivity.

The solid substrate fermentation is carried out using Penicillium brevicompactum.

The solid substrate for fermentation is selected from wheat bran, wheat rava, broken wheat, boiled rice, rice bran, rice rava, beaten rice, maize bran, maize grits, oat bran, bagasse, tapioca residue, soya grits, soya flakes, ceramic beads, glass beads, sponge or a mixture of one or more of these.

The nutrient feeding for fed-batch fermentation is done at the beginning of the fermentation.

The nutrient feeding for fed-batch fermentation is done throughout the fermentation.

The nutrient feeding for fed-batch fermentation is done at a constant or increasing rate across the course of fermentation.

The nutrient feeding for fed-batch fermentation is done with the carbon: nitrogen ratio in the feed maintained between 15: 1 to 100 : 1.

The nutrient feeding for fed-batch fermentation is done with the carbon: nitrogen ratio in the feed maintained between 30: 1 to 50 : 1.

The carbon feeds for fed-batch fermentation is selected from glucose, sucrose, starch (maize, wheat, tapioca, potato), modified starch, maltose, malto-dextrin, rava, soybean oil, acetate or a mixture of one or more of these.

The nitrogen feeds for fed-batch fermentation is selected from ammonium sulphate, ammonium nitrate, sodium nitrate, bacteriological peptone, yeast extract, casein hydrolyzate, soya peptone, soya flour, cotton seed flour, corn steep liquor or a mixture of one or more of these.

In the present invention, the nutrient feed has been designed in such a way that the culture does not experience substrate inhibition at any point of time. Initially the (product: substrate) yields were calculated based upon the carbon and nitrogen contents of solid substrate used for fermentation. Exhaust gas analysis of bio-reactor indicated that the production was growth-associated. Hence, the culture would

require both carbon (as carbon and energy source) as well as nitrogen for production. Various sources of carbon and nitrogen (with varying degree of solubility) were tried. The inhibitory and limiting levels of both carbon and nitrogen were established and finally a range of C: N ratio in the feed was arrived upon. Theoretical feed quantity can be increased up to a level just below the inhibitory level.

Thereafter, the feeding of balance amount of nutrients can continue as and when the feed gets finished. It has been found that the higher the feed, higher is the productivity.

The advantages of the current invention over the other reported methods are: (i) Easily scalable since the liquid feed is added directly (no need of spraying) to the solid substrate followed by mixing for uniform distribution (ii) Liquid feed can be easily steam sterilized (solid feed for intermittent addition needs sterilization by radiation) (iii) The feed is given intermittently which minimizes mixing (as compared to continuous fed-batch) and hence is ideal for shear sensitive cultures (iv) Overall dosing of nutrients per unit volume gets increased (higher the nutrients, higher the productivity) (v) Economical at large scale of operations.

The following Examples further illustrate the invention, it being understood that the invention is not intended to be limited by the details disclosed therein.

EXAMPLES EXAMPLE 1 The carbon and nitrogen sources were added to 10 g wheat bran in a petri plate keeping the C : N ratio from 5: 1 to 41 : 1. The amount of carbon fed was kept constant where as nitrogen was varied. The plates were inoculated with vegetatively growing Penicitlium brevicompactum inoculum and then incubated at 24 to 26 degree C for seven days. S. No. C: N Total Product in mg (Feed) per Plate 108 2 5:1 99.9 3 18:1 146.7 4 32 : 1 172. 8 5 41 : 1 311. 4 This table shows that C: N of 41 : 1 is most promising.

EXAMPLE 2 To a 10 g packing of wheat bran (WB) in petri plate, varying quantities of carbon and nitrogen feeds were given. The plates were inoculated with vegetatively growing Penicillium brevicornpactum inoculum and grown for 6 days. The temperature was controlled between 24 and 26 degree C. The results are shown below :

S. No. WB packing Qty. of feed Total product % Increase /plate (mL) (mg/plate) (9) 1 10 0. 0 108 (Control) 2 10 10. 0 106 - 1.8 3 10 13.0 144. 4 33.7 4 10 15.0 186.4 72.6 5 10 17.0 236.5 119 6 10 20. 0 274. 5 154 EXAMPLE 3 To a 100 g packing (height of 6.5 cm) of wheat bran in a 2 L jacketed glass vessel 190 mL of carbon and nitrogen feed was added at the time of inoculation with vegetatively growing Penicillium brevicompactum The temperature was controlled between 24 and 26 degree C for seven days. Air was sparged at a controlled rate across the bed in both the directions. Results are shown in the table below : S. No. Qty. of Feed Total Product % Increase (mL) (mg)

1 1440 I--. I 2 190 4050 181 EXAMPLE 4 To a 18 Kg packing (height of 6.5 cm) of wheat bran in a SS bioreactor, 26 Kg of carbon and nitrogen feed was added at the time of inoculation with vegetatively growing Penicillium brevicompactum.

The temperature was controlled between 24 and 26 degree C for seven days. Air was sparged at a controlled rate across the bed in both the directions. Results are shown in the table below : S. No. Qty. of Feed Total Product % Increase (Kg) (9) 1 - 121. 6- 2 26 295.0 143 EXAMPLE 5 To a 10 g packing of wheat bran in petri-plate, carbon and nitrogen sources were added before sterilization. After sterilization, the plate was inoculated with vegetatively growing Penicillium brez mpactum and grown for seven days. The temperature was controlled between 24 and 26 degree C. The results are shown in the table below: S. No Wheat bran Qty. of Qty. of Total Product % Packing Carbon nitrogen (mg) Increase

/Plate source source (g) (g) 1 10--180 0. 0 1 10 10 1.5 513.9 185.5 2 10 7.5 1.1 482.9 168.3 EXAMPLE 6 To a 10 g packing of wheat bran in petri-plate, 10 mL of carbon and nitrogen feed was added at the time of inoculation with vegetatively growing Penicillium brevicompactum. Afterwards, shots of nutrients were given ; plates were mixed and then again incubated at 24 to 26 degree C for another four days. The results are shown in the table below : S. No Wheat bran Initial Nutrient shot Total % Packing feed Qty. (Feeding Product Increase /Plate (mL) Day) (mg) (mL) 1 10-180 0. 0 2 10 20 - 342.0 90.0 3 10 10.0 10.0(3rd day) 378.5 110.3 4 10 5. 0 7. 5 (2nd day) 387. 0 115.0 7. 5 (4th day) 5 10 5. 0 5. 0 (2nd day) 397. 8 121. 0 10. 0 (4th day) EXAMPLE 7 To 75 g packing of ceramic beads in petri-plate, 10 mL or 5 mi of carbon and nitrogen feed was added at the time of inoculation with vegetatively growing Penicillium brevicompactum. ARerwards, shots of nutrients were given; plates were mixed and then again incubated at 24 to 26 degree C for another four days. The results are shown in the table below : S. No Ceramic Initial Nutrient shot Total % bead feed Qty. (Feeding Product Increase Packing (mL) Day) (mg) /Plate (mL) 1 75 - - 50 0.0 2 75 10 98. 0 96. 0 3 75 10 5.0(3rd day) 96.0 92.0 4 75 5.0 5.0(2nd day) 102.0 104.0 5.0(4th day)