Screening of nutrient parameters for mevastatin production by Penicillium citrinum MTCC 1256 under submerged fermentation using the Plackett-Burman design.

OBJECTIVE: Mevastain, an HMG-CoA reductase inhibitor produced by Penicillium citrinum. The Placket-Burman experimental design was used to identify the important nutrients influencing the production of mevastatin by Penicillium citrinum MTCC 1256 under submerged fermentation.
MATERIALS AND METHODS: Nine nutritional parameters, such as, glucose, glycerol, arrowroot, oats, urea, peptone, yeast extract, MgSO(4).7H(2)O, and CaCl(2).2H(2)O were screened by Placket-Burman experimental design in 12 experimental run.
RESULTS: Nine nutritional parameters, glucose, glycerol, arrowroot, oats, urea, peptone, yeast extract, MgSO(4).7H(2)O, and CaCl(2).2H(2)O contributed with 0.8114%, 24.0390%, 2.2786%, 0.1491%, 0.5608%, 47.5029%, 6.8092 %, 4.0980%, 10.5823% respectively towards mevastatin production by Penicillium citrinum under submerged fermentation.
CONCLUSION: Among the nine nutrient components glycerol, peptone, yeast extract, MgSO(4).7H(2)0, and CaCl(2).2H(2)O contributed to a large extent, urea had little impact, while glucose, oats, and arrowroot contributed moderately on production of mevastatin.

Among statins, mevastatin or compactin was the first statin investigated as a fungal secondary metabolite.[1] Mevastatin, a hypocholesterolemic molecule, is a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase, which is a regulatory enzyme for cholesterol biosynthesis, and also acts as an antifungal agent.[2-4] Mevastatin is also a precursor of pravastatin, which is also a hypocholesterolemic agent. Penicillium citrinum,[2356] P. cyclopium,[7] and Aspergillus terreus are among the few commercially used microbial strains used in the production of compactin.[8]

Designing of the fermentation medium is a critical and important process as the composition of the medium can significantly affect the product yield.[9] An optimally balanced culture medium is mandatory for the maximal production of secondary metabolites. Important medium variables can be screened by the Plackett-Burman experimental design.[10] It is a partial factorial design, where a large number of independent variables (N) are studied in a small number of experiments (N + 1).[11]

Materials and Methods

Microorganisms

Culture of Penicillium citrinum MTCC1256 was obtained from IMTECH Chandigarh, India, and was maintained on the slants of Potato-Dextrose Agar (PDA) medium at 4°C and subcultured at 30-day intervals.[1213]

Seed culture and fermentation

The culture of P. citrinum was grown on potato glycerol agar (PGA) slants, and the spore suspension (4 × 106 spores per mL) was prepared in glycerol water solution (15 g/l ). The inoculum media used in this study were glucose (20 g/l), glycerol (30 g/l), peptone (8 g/l), NaNO3(2 g/l), and MgSO4 (l g/l), which were dissolved in a water-soluble extract of soybean meal.[14] All fermentation experiments were carried out in 250 ml Erlenmeyer flasks containing 50 ml of production media, as per the experimental design. Each flask was inoculated at 5% v/v with spore suspension and incubated at 24°C and 220 rpm in an orbital shaker for 14 days.

Plackett-Burman experimental design

Glucose, glycerol, arrowroot, oats, urea, peptone, yeast extract, magnesium sulfate, and calcium chloride were the nine medium constituents selected for the study. The Plackett-Burman experimental design,[10] for 11 variables: nine nutritional components (independent variables) and two dummy variables [Table 1], were used, to evaluate the relative importance of various nutrients for mevastatin production in the submerged culture. In Table 2, each row represents a different variable.[15] For each nutrient variable two different concentrations high (+) and low (-) were tested [Table 2].

Table 1

Concentrations of variables at different levels in Plackett-Burman design for mevastatin production in submerged culture

Table 2

Plackett-Burman experimental design of 12 trials for 11 variables (9 nutrients + 2 dummies) along with the observed concentration of mevastatin in fermentation broth

Extraction of mevastatin

Mevastatin from fermentation broth was extracted according to the procedure given by Chakarvarti and Sahai, 2002b.[14] Fermentation broth (5 mL) was adjusted to pH 6.5 with either acid (H3PO4) or alkali (aq. NaOH). The broth was diluted five-fold with absolute ethanol, filtered through a 0.22 μm filter, and analyzed by High Performance Thin Layer Chromatography (HPTLC) (Camag, Muttenz, Switzerland)

Estimation of mevastatin

The extracted mevastatin were applied to the plates as 3-mm-width bands with a Camag 100 μL syringe, using a Linomat V (Camag, Muttenz, Switzerland) sample applicator on pre-coated silica gel aluminum plates 60F-254 (20 cm × 10 cm, with 0.2 mm thickness, E. Merck, Germany). The sample application rate kept at 150 nL/s was employed and the space between two bands was kept at 12.3 mm. Thin layer chromatography was performed using a mixture of toluene, ethyl acetate, and formic acid as the mobile phase, in the ratio of 3:2:1 per volume, in 10 × 10 cm twin through-glass chambers. Densitometric scanning was performed on a Camag TLC scanner III in absorbance mode, at 238 nm, for mevastatin, after drying in a hot air oven for 10 minutes at 100°C. The chromatograms were analyzed by WinCat software version 1.24.[16]

Data analysis of the Plackett-Burman design

The effect of each variable was determined according to the procedure given by Plackett-Burman.[17] The percentage of contribution of each nutrient parameter was calculated by using Design Expert 7.1 software of Stat ease Inc. USA.[15]

Results

Screening of the nutrient by the Placket-Burman experimental design for mevastatin production, using P. citrinum MTCC 1256, was carried out in this study. The purpose was to screen important nutritional variables. In this experiment, parameters of different mediums were tested for their nutritional ability, for mevastatin biosynthesis. Maximum mevastatin production was found in experiment trial 2 (589.3 mg/l), minimum in 5 (38.9 mg/l) [Table 2], under submerged fermentation. The effect of the dummy variables 1 and 2 were close to zero [Table 3], which indicates a successful experimental study. Experimental error was calculated and found to be 0.00005. Among the nine nutrient components used in the study, glycerol, peptone, yeast extract, MgSO4.7H2O, and CaCl2.2H2O had contributed to a large extent in mevastatin production by P. citrinum MTCC 1256. Urea and oats had little impact, while glucose and arrowroot contributed moderately [Figure 1].

Table 3

Effect of different medium components on mevastatin production

Figure 1

Percent contribution and effect of factors used in the Plackett-Burman design

Discussion

It has been found that glycerol is useful as a carbon source, it has a high contribution, and is effective in a lesser amount, as glycerol is a significant limiting factor influencing the biosynthesis of statin via growth.[18] Peptone has been found to be a better nitrogen source than yeast extract for mevastatin production by P. citrinum MTCC 11256. This is one reason why the growth of P. citrinum (biomass) largely depends on the type of nitrogen source. Moreover, a suitable concentration of peptone is important for a higher production of mevastatin; a lower biomass at lower peptone concentration results in less production of mevastatin.[18] In fungal nutrition, magnesium and calcium are noted as macronutrients and manganese, iron, copper, and zinc as micronutrients, but in the case of mevastatin production by P. citrinum, the contribution by magnesium and calcium is higher than that by iron and manganese.

Designing the medium is an open-ended, time-consuming, laborious process, involving a large number of experiments. The Plackett-Burman experimental design is a preliminary technique for the rapid illustration of effects of various medium constituents. It tests each variable at two levels only; hence, it cannot give an exact idea regarding the optimum level of the constituents required in a medium. Therefore, further optimization of selected nutrients, such as, glycerol, peptone, MgSO4.7H2O, CaCl2.2H2O, and KH2PO4 for mevastatin production by Penicillium citrinum, is necessary.