Production and Characterization of α-Galactosidase by a Multiple Mutant of Aspergillus niger in Solid-State Fermentation
Muhammad Siddique Awan1, Fatima Jalal2, Najma Ayub1, Muhammad Waheed Akhtar3 and Muhammad Ibrahim Rajoka2*
1Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, PK-54078 Islamabad, Pakistan
2Industrial Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, P.O. Box 577, Jhang Road, PK-38000 Faisalabad, Pakistan
3School of Biological Sciences, University of the Punjab, PK-54590 Lahore, Pakistan
Article history:
Received November 27, 2008
Accepted September 8, 2009
Key words:
enthalpy, entropy, production of α-galactosidase, Gibbs free energy, kinetics, solid-state fermentation
Summary:
α-Galactosidase is applied in the sugar industry to enhance sugar recovery from sugar beet syrup and to improve nutritional value of the soymilk. In the present investigation, the influence of process variables on the production of this important enzyme has been explored in a newly isolated multiple mutant strain of Aspergillus niger in solid-state fermentation (SSF). Defined fermentation parameters include substrate type (pure lactose and by-products of rice and flour mills as prime substrates), nitrogen source, incubation time, initial pH of the medium and incubation temperature. Extracellular α-galactosidase reached the value of 135.4 IU/g of dry substrate (IU/g) after 96 h of fermentation. Supplementation with 2 g of glucose and 3 g of corn steep liquor significantly increased the enzyme production, and maximum value of product yield (318 IU/g) by the mutant strain was significantly higher than that reported by the wild type (this work), or other A. niger mutants, recombinants and yeasts reported in literature as producers of elevated levels of α-galactosidase. Among three α-galactosidases, one possessing high subunit molecular mass proteins (99 and 100 kDa) has been characterized in both wild and mutant organisms. Thermal properties of the purified enzymes indicate that the mutation decreased the values of activation energy for the formation of enzyme-substrate (ES) complex, enthalpy, Gibbs free energy demand for substrate binding, and transition state stabilization. A thermodynamic study of irreversible inactivation of enzymes suggests that the mutant–derived enzyme is more thermostable than the native enzyme, which is attributable to amino acids involved in active catalysis. Because of these properties, the mutant organism is a novel organism and may be exploited for bulk production of thermostable α-galactosidase for the above industrial and nutritional applications.
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