Optimization of media composition for the production of cyclosporin A by Tolypocladium species

Transcription

1 Indian J Med Res 123, April 2006, pp Optimization of media composition for the production of cyclosporin A by Tolypocladium species K. Balaraman & Nisha Mathew Vector Control Research Centre (ICMR), Pondicherry, India Received May 25, 2005 Background & objectives: Cyclosporins are produced by certain species of the filamentous fungi, belonging to the genus Tolypocladium. While there are numerous reports on the use of cyclosporins in clinical studies, reports on the various aspects of their production have been very limited. Therefore, this study was carried to optimize the medium composition for the production of cyclosporin A, produced by a strain of the filamentous fungus, Tolypocladium species by static fermentation. Methods: The effect of different nutrients on the production of cyclosporin A, produced by Tolypocladium species in stationary culture was studied by growing the fungus for 21 days at o C under different media composition. Cyclosporin A was extracted by homogenizing the fungal cells with methanol and the cyclosporin A level was analyzed by high performance liquid chromatography (HPLC). Results: Among the six different media studied for the production of cyclosporin A, medium f containing glucose (8%), casein acid hydrolysate (3%), malt extract (2%), peptone (1%) and DLα-amino butyric acid (0.5%) favoured the maximum production ( g/l medium or g/kg biomass). Interpretation & conclusion: This study showed that by optimizing the composition of fermentation media enhanced production of cyclosporin A was obtained. Since the strain Tolypocladium (VCRC F21 NRRL No.18950) produces a high level of cyclosporin A in the identified fermentation medium, it could be exploited for industrial production. Key words Biomass - cyclosporin A - immunosuppressive agent - static fermentation - Tolypocladium Cyclosporins are produced by certain species of the filamentous fungi, belonging to the genus Tolypocladium 1. While there are numerous reports 525 on the use of cyclosporins in clinical studies, reports on the various aspects of production of these have been very limited. The production of cyclosporin in

2 526 INDIAN J MED RES, APRIL 2006 submerged fermentation has been reported to vary with respect to the production strain 2-4, fermentation conditions 5-12, and the nutrient composition of the culture medium. Most of these reports indicate that the production of cyclosporins per litre of the production medium is not up to appreciable level. In the present study production of cyclosporin A was attempted by static fermentation using different culture media and the production strain Tolypocladium species 16. Material & Methods Tolypocladium sp. (VCRC F21 NRRL No.18950) obtained as lyophilized conidia from the culture collection of Vector Control Research Centre, Pondicherry was used. The conidia were suspended in 1 ml of culture maintenance medium [2% malt extract, 0.4% yeast extract and 2% agar in sterile water (ph 5.2)] and using a standard loop the suspension was inoculated to agar slopes 16. The agar slopes were incubated for 14 days at 25 o C. The culture on agar slopes was transferred to 50 ml of the conidia production medium [dextrose (2%), peptone (1%) and casein acid hydrolysate (1%) in sterile water (ph 6.00)] and incubated on a rotary shaker at 150 rpm and 25 o C for 4 days (first stage inoculum). The first stage inoculum was transferred to 200 ml conidia production medium in 1000 ml conical flask and incubated at 110 rpm and 25 o C for 3 days (second stage inoculum). Twenty Erlenmeyer flasks (500 ml capacity) containing 50 ml of each production medium (Table) were inoculated with second stage inoculum (10% v/v) and incubated for 21 days under static conditions at o C. Two sets of media were inoculated. The first set had glucose and glycerol as carbon source each at 4 per cent (w/v) level. The second set had glucose alone as carbon source at two levels (4 and 8% w/v). Three nitrogen sources viz., casein acid hydrolysate (3%), malt extract (2%) and peptone (1%) in different combinations were tried. The precursor of cyclosporin A DL-α- amino butyric acid was added to all the media at the concentration of 0.5 per cent (w/v). All experiments were done in duplicate. After 21 days the fungal biomass was harvested from the culture flasks by decanting the spent broth, the fungal biomass was blot dried using country filter paper, the wet weight was determined and then homogenized in a blender. The homogenate was extracted thrice with methanol in the ratio of 1:2 (w/v) by keeping over a rotary shaker at 250 rpm for 1 h and filtering through whatmann no.1 filter paper. The filtrates (crude extracts) were pooled and the total volume was determined. The cyclosporin A level in the crude extract was analyzed by HPLC (Shimadzu, Tokyo, Japan) HPLC model LC-3A equipped with CTO-6A column oven, SPD-2A variable wavelength UV detector, and CR- 3A integrator) as per the method reported 17 earlier with a slight modification. The analysis was done using a Phenomenex C8 (California, USA) (5cm X 4.6 mm ID, 5 µm particle size) column at 60 o C with acetonitrile water (70:30) containing 0.01 per cent orthophosphoric acid (H 3 PO 4 ) as mobile phase at a flow rate of 1 ml/min with UV detection at 210 nm. The samples for analysis were prepared by removing the solvent from 1 ml of each pool of the crude extract. The residues were redissolved in HPLC grade acetonitrile (1 ml) and 5 µl of each sample was injected into HPLC. The retention time and peak area were compared with the standard cyclosporin A (Sandimmune, Sandoz) to get the level of cyclosporin A in the crude extracts.

3 BALARAMAN & MATHEW : PRODUCTION OF CYCLOSPORIN A BY TOLYPOCLADIUM SP. 527 Statistical analysis of the data was done by Oneway ANOVA using SPSS (Ver. 11.0) software. production of cyclosporin A in the medium b ( g/l medium or g/kg biomass). Results & Discussion The retention time of cyclosporin A was found to be min. Six different media (Table) were used for the production of cyclosporin A. The production of biomass from different media varied between and g/l and that of cyclosporin A (crude level) varied between and g/l of the production medium or and g/kg of biomass (Fig. 1). The maximum biomass was obtained from medium e followed by d. The maximum volumetric production of cyclosporin A ( g/l medium) or ( g/kg biomass) was obtained with the medium f where in the carbon source was glucose (8%). The volumetric production of cyclosporin A was less in the medium e (with glucose at 4% level) ( g/l medium or g/kg biomass). In the medium d with glucose and glycerol each at 4 per cent level enhanced level of production was observed ( g/l medium or g/ kg biomass) which was comparable with that obtained in medium a ( g/l medium or g/kg biomass) 16. In the absence of peptone, there was a drastic reduction in the The level of cyclosporin A produced with medium f at different intervals of incubation is given in Fig. 2. The production of cyclosporin A was slow for the first half of the incubation period followed by a rapid increase in the second half. The production was maximum on day 21 of the incubation period and after that lysis of the fungal mycelium has set in. Cyclosporin being an intracellular metabolite would be released into the culture medium if the fungus is permitted to grow beyond 21 days due to autolysis of the fungal cell wall. In the case of biomass, a rapid increase was observed in the first half which slowed down in the second half of the incubation period (Fig. 2). The present study showed that the medium containing glucose (8%), casein acid hydrolysate (3%), malt extract (2%), peptone (1%) and DL-αamino butyric acid (0.5%) favoured the maximum production of cyclosporin A by Tolypocladium sp. (VCRC F21 NRRL No.18950) under static fermentation which was significantly (P<0.05) different from that produced by medium a containing glucose (4%), glycerol (4%), casein acid hydrolysate (3%), malt extract (2%), peptone (1%) and DL-α-amino butyric acid (0.5%). Table. Media composition used for the production of cyclosporin A Medium Carbon source (%) Nitrogen source (%) code Glucose Glycerol Casein acid Malt Peptone hydrolysate extract a b c d e f

4 528 INDIAN J MED RES, APRIL 2006 Biomass yield (g/l) Cyclosporin A yield (g/l) Cyclosporin A yield (g/kg biomass) Fig. 1. Production of biomass and cyclosporin A in different culture media (values are mean + SD of 2 observations).

5 BALARAMAN & MATHEW : PRODUCTION OF CYCLOSPORIN A BY TOLYPOCLADIUM SP. 529 Production of cyclosporin A (g/l) Production of biomass (g/l) Incubation period (days) cyclosporin A Biomass Fig. 2. Production of cyclosporin A (VCRC F21 NRRL No.18950) at different intervals of incubation (values are mean + SD of 2 observations). Earlier the production of cyclosporin A with wheat bran 9 as the solid substrate was attempted using Tolypocladium sp (VCRC F21 NRRL No.18950) and it was found that the production (1.98 g/kg bran) was several times more than that obtained (0.08 g/l medium) from submerged fermentation with medium containing 2 per cent glucose, 1 per cent casein acid hydrolysate, 1 per cent peptone and 1 per cent malt extract 10. Immobilization of the fungus VCRC F21 in calcium alginate in a packed bed reactor under batch and continuous flow modes could yield cyclosporin A up to 680 mg/l medium 11. In the present study, a variety of carbon and nitrogen sources at different combinations were tried for enhancing the production of cyclosporin A by the Tolypocladium sp. (VCRC F21 NRRL No.18950) under static fermentation. It was observed that a combination of glucose (8%), casein acid hydrolysate (3%), malt extract (2%), peptone (1%) and DL-α-amino butyric acid (0.5%) favoured higher production of cyclosporin A compared to the production reported earlier in submerged and solid state fermentations. Acknowledgment The authors thank Dr P.K. Das, Director, Vector Control Researh Centre for providing the necessary facilities and Shri S. Venugopalan for technical assistance. References 1. Dreyfuss M, Harri E, Hoffman H, Kobel H, Pache W, Tscherter H. Cyclosporin A and C, new metabolites from Trichoderma polysporum (Link ex Pers.) Rifai. Eur J Appl Microbiol Biot 1976; 3 : Agathos SN, Parekh R. Enhancement of cyclosporin production in a Tolypocladium inflatum strain after epichlorohydrin treatment. J Biotechnol 1990; 13 :

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