Effect of C/N ratio and microelements on nutrient dynamics and cell morphology in submerged fermentation of Aspergillus giganteus MTCC 8408 using Taguchi DOE.

This paper is concerned with the effect of individual Na+, K+, Mg2+ and Ca2+ ions upon the nutrient dynamics and cell morphology during submerged fermentation of Aspergillus giganteus MTCC 8408 in a medium containing ample soluble starch, corn steep liquor and proteose peptone supply. Nutrient dynamics was elucidated using Taguchi's DOE (design of experiment) L8 orthogonal array (OA) with carbon, nitrogen and four most influensive microelements at their assigned ratio namely; K+/Ca2+ and Mg2+/Na+. Scanning electron microscopy (SEM) was used to correlate the effect of selected factors on morphological changes and nutrient dynamics during submerged fermentation. At higher K+/Ca2+ ratio 4.78:1; the maximum growth was figured out in terms of dry weight, while the inhibition of dry weight production was observed at higher Mg2+/Na+ ratio 0.94:1 with enhanced intracellular afp production. At lower C/N ratio 27.4:1; filamentous form of growth was maintained with the hyphae being scarcely branched without bulbous cells. Membrane perturbation due to the induction of intracellular oxidative stress was noticed at higher C/N ration. Taguchi DOE resulted in afp yield per substrate utilized, Y p/s of 1.08 mg g-1 soluble starch utilized with gram dry cell weight (gdcw) of 23.9 g L-1 while afp yield per biomass utilized resulted, Y p/x of 1.12 mg afp gdcw-1 L-1 and biomass yield per substrate utilized, Y x/s of 1.195 gdcw g-1 L-1. The present study revealed an opportunity to develop a cost effective methods for cell propagation so as to yield effective inoculum levels towards mass production and formulation studies.

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

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.

Molecular docking and dynamics simulations of A.niger RNase from Aspergillus niger ATCC26550: for potential prevention of human cancer.

The aim of the present research was to study the anticancer effects of Aspergillus niger (A.niger) RNase. We found that RNase (A.niger RNase) significantly and dose dependently inhibited invasiveness of breast cancer cell line MDA MB 231 by 55 % (P<0.01) at 1 µM concentration. At a concentration of 2 µM, the anti invasive effect of the enzyme increased to 90 % (P<0.002). Keeping the aim to determine molecular level interactions (molecular simulations and protein docking) of human actin with A.niger RNase we extended our work in-vitro to in-silico studies. To gain better relaxation and accurate arrangement of atoms, refinement was done on the human actin and A.niger RNase by energy minimization (EM) and molecular dynamics (MD) simulations using 43A(2) force field of Gromacs96 implemented in the Gromacs 4.0.5 package, finally the interaction energies were calculated by protein-protein docking using the HEX. These in vitro and in-silico structural studies prove the effective inhibition of actin activity by A.niger RNase in neoplastic cells and thereby provide new insights for the development of novel anti cancer drugs.