Samina Khan, Saima Saleem and Abid Azhar
Department of Biochemistry, University of Karachi, Karachi, Pakistan
Introduction:Aspergillusspecies are highly abundant fungi worldwide involved in the production of cell wall-degrading enzyme, pectinase. Pectinase is a heterogeneous group of enzymes that have biotechnological, functional and biological applications in food processing, fruit ripening and textile industries. The recovery of by-products from agri-food industry is currently one of the major challenges of biotechnology. In the present study, an indigenous fungal strain identified asAspergillus niger-FS001 was isolated from moldy vegetables and fruits samples that showed maximum pectinase production.
Materials & Methods:Submerged fermentation technique was used for pectinase production from fungal strain Aspergillus niger-FS001. The microscopic features of the strains were studied by Lacto Phenol Cotton Blue staining technique. Lowry’s method was used to determine the total protein. The enzyme assay was done by using citrus pectin and galacturonic acid monohydrate. The amount of reducing sugar released was analyzed by 3, 5-dinitrosalicylic acid method. Partial purification of pectinase was carried out by gradient ammonium sulphate precipitation. Extracellular pectinase was purified by gel filtration chromatography. Molecular weight of pectinase was determined by SDS-PAGE and zymography.
Results: The maximum enzyme production was obtained after 120 hours. The optimum temperature and pH for maximum enzyme production by Aspergillus niger-FS001 were 30°C and 5.5, respectively. Carbon source, such as pectin, enhanced the maximum production of enzyme. Simultaneously, the combination of yeast extract, peptone, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and calcium chloride in optimized concentrations also enhanced the enzyme production. Partial purification of pectinase was achieved by 40% ammonium sulphate for maximum precipitation of enzyme. The enzyme was purified to homogeneity with 95.94 fold purification. The molecular weight of purified pectinase was found to be 129 kDa by SDS-PAGE. Zymography of the purified enzyme was also performed, which confirmed the homogeneity of the purified enzyme.
Conclusion: The maximum economical production of pectinase by AspegillusnigerFS001 was achieved by utilizing cheap indigenous substrate that has potential use in several biotechnological industries.
Keywords: Pectinase, Aspergillus niger, enzymes, pectin, biotechnological industries.