Production, Purification and Characterization of Polygalacturonase from Aspergillus niger in Solid State and Submerged Fermentation Using Banana Peels

摘要

Aims: Pectinases are extracellular enzymes produced by microorganisms which break down pectic polysaccharides of plant tissues into simpler molecules like galacturonic acids. Polygalacturonase catalyzes hydrolysis of α-1,4-glycosidic linkages in polygalacturonic acid producing D-galacturonate. Polygalacturonases are hydrolytic depolymerases with endo and exo activities that are widely used in food and chemical industries. This present study aimed to isolate fungal strain capable of producing polygalacturonase, compare its production in SSF and SmF, optimize the cultural conditions as well as purify and characterize the enzyme. Study Design: The design adopted to evaluate the influence of cultural conditions on the enzyme production and physicochemical parameters on purified enzyme activity was One-Factor-at-a-Time approach (OFAT). Place and Duration of Study: Department of Microbiology, Faculty of Science, University of Port Harcourt, Nigeria, between November 2014 and December 2015. Methodology: A total of 12 fungal strains were isolated from fresh banana peels. The isolates were screened for polygalacturonase producing ability using conventional methods and the isolate with the highest zone of inhibition was selected for further studies. The production of polygalacturonase by Aspergillus niger in solid state and submerged fermentation using banana peels as carbon source were compared and the best was used for further production studies. The effect of cultural conditions on polygalacturonase production by the fungus was evaluated. The crude enzyme was purified using ammonium sulphate precipitation and gel filtration on Sephadex G100 and effect of some physicochemical parameters on purified enzyme activity was also determined using standard methods. Results: A total of 5 out of 12 fungi isolated from fresh banana peels were found to be pectin degraders. The most efficient isolate was identified as Aspergillus niger based on its colonial, morphological and microscopic examination. Solid state fermentation (SSF) was found to be more suitable (60.20%) for polygalacturonase production by Aspergillus niger compared to the submerged fermentation (SmF) (39.80%). Optimization of process parameters revealed that 48 h of incubation was the optimum for polygalacturonase production in solid state fermentation, while 72 h was observed for submerged fermentation. Supplementation of fructose to the fermentation medium led to increased enzyme production. KNO₃ was the best nitrogen source for polygalacturonase production. The enzyme was purified by ammonium sulphate precipitation (75%) and gel filtration on Sephadex G100. The purified polygalacturonase showed a specific activity of 166.67U/mg with 8.59% yield and purification fold of approximately 42. The optimum temperature for polygalacturonase activity was 40°C. The purified enzyme was stable within 20-50°C for 1 h. The enzyme has an optimum pH activity at 5.0 and was stable within the pH range of 4-6. Co2+ strongly stimulated the enzyme activity while Ba2+ showed the highest inhibitory effect on the enzyme activity. Conclusion: This study has revealed an enhanced production of polygalacturonase by A. niger under solid state fermentation using cost effective agricultural waste (banana peels). Thus, the re-utilization of banana peels as substrate for production of the enzyme by the fungus will minimize the pollution problems their presence may pose to the environment. In addition, the results obtained in this study indicated that the polygalacturonase from A. niger could found immense potential application in industrial sectors and biotechnology.