An investigation on the production, partial purification, characterization of inulinase enzyme and mathematical modeling of its fermentations

The objectives of this study were to determine the best carbon source for enzyme production, to optimize the medium formulation used in enzyme production, to make the kinetic modeling of the fermentations, to purify partially the produced enzyme, to determine the properties of the enzyme, and to model mathematically the fermentations and substrate specificity. According to the results obtained; among the ten different carbon sources used in the production of Aspergillus niger A42 (ATCC 204447) inulinase (Iase), molasses was the best (383.73 U / mL). The optimum working conditions of the enzyme were pH 4.8, 60ºC and 10 min incubation time. In the optimization of the medium composition with Plackett-Burman Design (PBD), the optimum medium consisted of 1% yeast extract and 1% peptone (Iase=1011.02 U/mL, sucrase (Sase)=834.28 U/mL and I/S ratio=1.22). The concentration of yeast extract and peptone used in the medium was optimized by Central Composite Design (CCD) and the optimum medium composition consisted of 4.2% yeast extract (Iase=1294.50 U/mL, Sase=1076.85 U/mL, I/S ratio=1.20). As a result of kinetic modeling, the enzyme production in PBD-optimized medium is non-growth-associated (α≤0 and β≠0). In CCD-optimized medium, the enzyme production is mixed-growth-associated (a≠0 and β≠0). Fermentations in the small-scale bioreactor (5-L) should be carried out in a pH-uncontrolled and aerated environment (Iase=1810.76 U/mL, Sase=1537.88 U/mL and I/S ratio=1.18). As a result of fermentation in large scale bioreactor (30-L), Iase, Sase and I/S ratio were determined as 791.35 U/mL, 616.90 U/mL and 1.28, respectively. The enzyme solution obtained was partially purified by centrifuge-time combination (16873 g-5 min, Iase=1181.18 U/mL, purification coefficient (PC)=1.39) and UF membrane (10 kDa, Iase=12065.20 U/mL, PC=5.33). On the other hand, in the substrate specificity of the enzyme, the inulin affinity of the low-activity enzyme was found to be higher than sucrose (Km inulin=17.79 g/L < Km sucrose=49.43 g/L). In contrast, the sucrose affinity of the high-activity enzyme was found to be higher than inulin (Km inulin=28.77 g/L > Km sucrose=25.95 g/L). It was determined that activity increased with the increasing amount of enzyme added on substrate. Mn2+ was the activator of the enzyme whereas Cu2+ and Ag+ were inhibitors of the enzyme. It was determined that the enzyme produced was thermostable at 30ºC, 40ºC and 50ºC. In addition, thermal inactivation and thermodynamic parameters of the enzymatic reaction were also calculated. Hereunder, the Ea, Eia, and Z values were computed as 37.3 kJ/mol.K, 112.86 kJ/mol, and 12.80ºC, respectively. When the temperature was increased from 60ºC to 80ºC, the inactivation rate constant and entropy of the enzyme increased while half-life, D-value, enthalpy and free energy decreased. Besides, it was determined that the molecular weight of the enzyme was between 60 kDa and 70 kDa. The experimental data of fermentations performed in the medium composition optimized by PBD and CCD were estimated using mathematical models. On the other hand, the experimental kinetic parameters were also calculated and compared using the predicted values from the models. Finally, the experimental data of the substrate specificity of the enzyme were also predicted using mathematical models. Experimental Vmax and Km values were compared with Vmax and Km values estimated from the models. The best model(s) was/were determined according to the model comparison data (root-mean-square-error (RMSE), mean-absolute-error (MAE), mean standard deviation (MSD), regression coefficient (R2), slope (m), Akaike's information criterion (AIC), bias factor (BF), and accuracy factor (AF)). Consequently, the production of inulinase enzyme, kinetic modeling of the fermentations, partial purification and characterization of the produced enzyme and mathematical modeling of the fermentations and the substrate specificity have been performed successfully. The study includes both upstream processing and downstream processing.