A two-step optimization procedure using central composite design with four factors (concentrations of maltrin and corn steep liquor (CSL), agitation speed and inoculation ratio) was used in order to investigate the effect of these parameters on the polygalacturonase (PG) enzyme activity, mycelia growth (biomass) and morphology (pellet size) of Aspergillus sojae ATCC 20235. According to the results of response surface methodology (RSM), initial concentrations of maltrin and CSL and agitation speed were significant (p < 0.05) on both PG enzyme production and biomass formation. As a result of this optimization, maximum PG activity (13.5 U/ml) was achievable at high maltrin (120 g/l), at low CSL (0 g/l), high agitation speed (350 rpm) and high inoculation ratio (2 × 10 7 total spore). Similarly, maximum biomass (26 g/l) could be obtained under the same conditions with only the difference for higher level of CSL requirement. The diameter of pellets in all optimization experiments ranged between 0.05 and 0.76 cm. The second optimization step improved the PG activity by 74% and the biomass by 40%.

a b s t r a c tCrude exo-polygalacturonase enzyme (produced by Aspergillus sojae), significant for industrial processes, was characterized with respect to its biochemical and thermal properties. The optimum pH and temperature for maximum crude exo-polygalacturonase activity were pH 5 and 55°C, respectively. It retained 60-70% of its activity over a broad pH range and 80% of its initial activity at 65°C for 1 h. The thermal stability study indicated an inactivation energy of E d = 152 kJ mol À1. The half lives at 75 and 85°C were estimated as 3.6 and 1.02 h, respectively. Thermodynamic parameters, DH * , DS * and DG * , were determined as a function of temperature. The kinetic constants K m and V max , using polygalacturonic acid as substrate, were determined as 0.424 g l À1 and 80 lmol min À1, respectively. SDS-PAGE profiling revealed three major bands with molecular weights of 36, 53 and 68 kDa. This enzyme can be considered as a potential candidate in various applications of waste treatment, in food, paper and textile industries.

The effect of solid substrates, inoculum and incubation time were studied using response surface methodology (RSM) for the production of polygalacturonase enzyme and spores in solid-state fermentation using Aspergillus sojae ATCC 20235. Twostage optimization procedure was applied using D-optimal and face-centered central composite design (CCD). Crushed maize was chosen as the solid substrate, for maximum polygalacturonase enzyme activity based on D-optimal design. Inoculum and incubation time were determined to have significant effect on enzyme activity and total spore (p < 0.01) based on the results of CCD. A second order polynomial regression model was fitted and was found adequate for individual responses. All two models provided an adequate R 2 of 0.9963 (polygalacturonase) and 0.9806 (spores) (p < 0.001). The individual optimum values of inoculum and incubation time for maximum production of the two responses were 2 × 10 7 total spores and 5-6 days. The predicted enzyme activity (30.55 U/g solid) and spore count (2.23 × 10 7 spore/ml) were very close to the actual values obtained experimentally (29.093 U/g solid and 2.31 × 10 7 spore/ml, respectively). The overall optimum region considering the two responses together, overlayed with the individual optima. Solid-state fermentation provided 48% more polygalacturonase activity compared to submerged fermentation under individually optimized conditions.

A full factorial statistical design, with the factors of, two taxonomically different strains, seven types of seed culture formulations (slants) and two types of fermentation media were used to investigate the effect of these parameters on the morphology and polygalacturonase production. The rheology of the final fermentation medium was analyzed and appropriate mathematical model was applied to calculate suspension viscosity. It was found that most fermentation broths showed non-Newtonian flow behavior. According to statistical analysis, factors of strain types and fermentation media and the interaction between them were found significant on the enzyme activity. The effect of seed culture formulations (slants) were found insignificant at the significance level of 1%. Interaction of slants with strain types and fermentation media were also found insignificant. Considering the morphology of the final culture, Aspergillus sojae with the desired pellet morphology in a complex media, inoculated with a seed culture prepared from molasses resulted in maximum polygalacturonase enzyme activity (0.2 U/ml) and lowest suspension viscosity with a broth rheology close to Newtonian flow behavior.

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