Optimization and partial characterization of intracellular anticandidal protein from Aspergillus giganteus MTCC 8408 using taguchi DOE

Abstract

A new intracellular antifungal protein (afp) production with average molecular weight 24.3 kDa and yield of 0.65 ± 0.1 mg/gram dry cell weight (gdcw) of mycelia in submerged fermentation of Aspergillus giganteus MTCC 8408 was optimized. Taguchi's DOE (design of experiment) L₂₇ orthogonal array (OA) was constructed using Qualitek-4 software with 8 most influensive factors namely, culture pH, temperature, slant age, inoculum volume, agitation and KH₂PO₄. Scanning electron microscopy (SEM) was used to correlate the effect of selected factors on fungal cell morphology and afp production. The crude protein purification was accomplished using pure ammonium sulfate fractionation followed by carboxymethyl cellulose (CMC) ion-exchange chromatography and sephadex G-100 gel filtration. The average molecular mass of the purified protein was figured by silver stained SDS (sodium dodecylsulphate)-PAGE (poly-acryl amide gel electrophoresis). In vitro antifungal susceptibility assay was profiled against Candida albicans NCIM 3471 and minimum inhibitory concentrations (MICs) were in the range 3 to 4 µg/ml. Characterization of protein was observed with FTIR (Fourier transform infrared spectroscopy) analysis. The optimal production condition for crude afp was obtained as follows: soluble starch: 20 g/l; Corn steep liquor (CSL, 2%) + proteose peptone (PP, 1%): 30 g/l; pH: 5.8; temperature: 25°C; slant age: 3 d; inoculum size: 5% (v/v); agitation: 180 rpm; KH₂PO₄: 0.1 g/l. The validation experiments using optimized conditions confirmed an improvement in afp production by 59.4% against the expected enhancement of afp production by 61.22%. The present statistical optimization study revealed an opportunity to promote economical design at the industrial level for future scale up of effective antifungal agent against opportunistic oral and vaginal infection.

Keywords: Antifungal protein (afp); Aspergillus giganteus MTCC: 8408; Candida albicans NCIM 3471; Characterization; Minimum inhibitory concentration (MIC); Taguchi (DOE) L27 orthogonal array (OA).

Figure 1.

Impact of selected factor levels on afp production by A. giganteus MTCC: 8408 under submerged fermentation.

Figure 2.

Morphological behavior of Aspergillus giganteus MTCC 8408, showing the effect of various nutrients composition on optimization. Lateral resolution: 2–10 µm; magnification: 5KX; EHT: 18.00 KV; WD: 9.5 mm.

Figure 3.

Relative influence of factors and interactions.

Figure 4.

Performance distribution plot: current vs. improved condition.

Figure 5.

Silver stained SDS-PAGE of Aspergillus giganteus MTCC 8408 intracellular protein taken at 4 different fractionations (0–30%, 30–50%, 50–70%, 70–90%).

Figure 6.

FTIR analysis of Aspergillus giganteus MTCC 8408 intracellular protein.