Optimizatıon of microbial polygalacturonase production using different media compositions and fermentation strategies

In recent years, the evaluation of low-cost materials in fermentation media to reduce production costs has been an approach adopted by researchers. In this study, 11 different carbon sources and 9 different nitrogen sources were used in the production of polygalacturonase enzyme from Aspergillus sojae, and bioreactor experiments were carried out with the most efficient ones. To control excessive fungal growth in fermentation media, microparticle technique was used and thus repeatable results were obtained in all trials. In a small-scale bioreactor, the concentration of substrate and aeration rate are optimized using the Central Composite Design for the highest enzyme activity. For this purpose, after ultrafiltration, the highest enzyme activity was determined as 126.91 U/ml for the fermentations performed under optimum conditions (54.14 g/L substrate concentration, 0.25 vvm aeration rate). Enzyme production was performed in small (5-L) and large-scale (30-L) bioreactors using the determined optimum conditions and the compatibility of the experimental results with 10 different mathematical models was investigated. The most compatible models with experimental data for small-scale bioreactor trials were determined to be Modified Gompertz, Modified Richards, Modified Logistics and Huang models. For large-scale production, activity estimates closest to experimental data were calculated with Modified Richards and Modified Logistics models. Broth samples obtained from fermentations were passed through an ultrafiltration system using the membranes of 10 and 100 kDa as cut-off value for partial purification of the enzyme. Crude enzyme and ultrafiltered solutions were analyzed for quantification of total protein, and thus specific activity values were determined. Lyophilization (freeze-drying) process was implemented to the permeate and retentate samples obtained from ultrafiltration to determine the potential of the enzyme to be produced as a commercial preparation, and its purification coefficients were calculated. The affinity of the produced polygalacturonase to the substrates of pectin, carboxymethyl cellulose, and polygalacturonic acid was examined, and Km values were calculated as 21.2, 10.8 and 4.1 mg/ml, respectively. Further, Vmax values of the enzyme in the substrates of pectin, carboxymethyl cellulose, and polygalacturonic acid were determined as 101.7, 33.7 and 40.2 µmol min−1 mg−1, respectively. It was found that EDTA agent, Ba2+, Fe3+, Mn2+, Co2+ and Sr2+ ions have an inhibitory effect on the enzyme, although the produced polygalacturonase was stabile during interaction of the enzyme with Ag+, K+ and Na+ ions. Finally, basic a cost analysis was prepared based on the current unit prices of raw materials, consumables and energy expenditures for upstream and downstream processing. In the fermenter, operation conditions that are difficult to control due to excessive fungal growth were improved with the use of microparticles and standard-repeatable productions were achieved. The compatibility of production data with the mathematical models was determined. Polygalacturonase was successfully characterized, partially purified and lyophilized. In addition, an economic analysis was prepared for the fermentation performed with natural carbon sources to show the low-cost production potential of the polygalacturonase.