Characterization of Glucoamylase Produced by Aspergillus niger and Rhizopus sp.
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Date
2015-04-20
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Abstract
Amylase enzymes are important enzymes employed in starch processing industries for hydrolysis of polysaccharides into simple sugars. Microorganisms including a number of fungal species have been used to produce amylases more economically than from other sources. Glucoamylase (C3009H4570N782O1012S13) is an exoenzyme that removes glucose units consecutively from the nonreducing ends of starch and oligosaccharides. The enzyme also cleaves α-1, 6- and α-1, 3-bonds but at a slower action. Glucoamylase is used in processed-food industry, fermentation technology, textile and paper industries. In this study, four native fungal isolates, Aspergillus niger, Aspergillus flavus, Rhizopus species and Fusarium oxysporum and the potential of five solid substrates, wheat bran, rice bran, groundnut pod, maize bran and cocoa pod for glucoamylase production were investigated using Solid-State Fermentation process. Isolates of Aspergillus niger and Rhizopus species on wheat bran as substrate at spore concentrations of 1 x 107 per ml produced the highest enzyme activities under optimum growth conditions. Glucoamylase production was found to be affected by temperature, pH, incubation period, nature of substrate and the kind of microorganism used. Glucoamylase production by Aspergillus niger was found to be affected by nitrogen. Glucoamylase produced by Aspergillus niger yielded maximum enzyme activity of 6.66 U/ml in 18 hours of incubation period at a temperature of 40ºC, nitrogen concentration of 0.2 g/l and at pH 5.0. Similarly, glucoamylase produced by Rhizopus species gave maximum enzyme activity of 4.44 U/ml in 18 hours of incubation period at a temperature of 40ºC and pH 4.5. Molecular weights of proteins in culture filtrates were determined by SDS-PAGE. Proteins with molecular weights 61.48, 29.68, 21.06 and 12.33 KDa were identified from culture filtrates of Aspergillus niger and proteins with molecular weights 96.40, 65.56, 51.80, 29.05 and
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19.75 KDa were found from culture filtrates of Rhizopus species. Kinetic studies using Hanes-Woolf’s plot and starch as substrate gave Kmax = 0.0009548 g/l and Vmax = 2.387 g/l.min for enzyme produced by Aspergillus niger and Kmax = 0.0007443 g/l and Vmax = 2.481 g/l.min for enzyme produced by Rhizopus species
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A thesis submitted to the
School of Graduate Studies, Kwame Nkrumah University of Science and Technology
in partial fulfillment of the requirement for the degree of
DOCTOR OF PHILOSOPHY
Department of Chemical Engineering, College of Engineering.