Metabolic engineering and thermodynamic characterization of an extracellular β-glucosidase produced by Aspergillus niger

Abstract

The production of an extracellular β-glucosidase by Aspergillus niger NRRL 599 was optimized using submerged fermentation technique. Effect of different media, different carbon sources, initial pH of the fermentation medium, temperature, incubation period and inoculum size on the production of β- glucosidase enzyme was investigated. A. niger NRRL 599 produced maximum extracellular β- glucosidase (4.48 U/mg) in Eggins and Pugh medium with 1% wheat bran (w/v) at pH 5.5 inoculated with 4% conidial suspension after 96 h of incubation at 30°C. Purified β-glucosidase gave K_m and V_max values of 3.11 mM and 20.83 U/mg respectively for pNPG hydrolysis. The enzyme was optimally active at pH 4.8 and at temperature of 60°C. Thermodynamic parameters, E_a, ΔH and ΔS were found to be 52.17 KJ/mol, 49.90 J/mol.K and -71.69 KJ/mol, respectively. The pKa1 and pKa₂ of ionizable groups of active site residues involved in V_max were calculated to be 4.1 and 6.0 respectively.

Key words: β-Glucosidase, Aspergillus niger, kinetics, thermodynamics.