GENETIC MANIPULATION OF ASPERGILLUS NIGER FOR HYPER-PRODUCTION OF AND GALACTOSIDASES

α- and Galactosidase production from different carbon sources by using Aspergillus niger, and its DG-resistant mutant derivative was studied in solid state fermentation. Production of the enzymes was enhanced by ray mutagenesis and mutant strain was selected on 1 % (w/v) deoxy-D-glucose (DG). Optimization studies on carbon and nitrogen sources, initial pH of the Vogels medium and fermentation temperature were performed. Both strains of Aspergillus niger grown on fourteen different carbon sources revealed that the enzymes were formed and released with different levels and varied with the type of carbon sources. Distinct variations in the values of µ, Qs, Yx/s and Qx of parent and mutant cultures on different carbon sources were noted. The highest level of (1- and β-galactosidase production was recorded with wheat bran (58.86 IU/I/h, 176.3 IU/l/h for mutant as compared to 29.77 IU/l/h, 83.1IU/l/h for parent, respectively). Substantial amounts of enzymes were also obtained with rice bran, rice polishing and sugarcane bagasse. Among the five tested nitrogen sources, corn steep liquor produced the highest amounts (2-fold increase) of (α-and β-galactosidase (56.78 IU/l/h, 168.0 IU/l/h). The mutant strain produced 127.15 IU/I/h and 371.4 IUIl/h of (α- and β-galactosidase activity, which were significantly higher than those recorded from parent strain of Aspergillus niger. Studies revealed that the enzymes produced optimally at initial pH of 5.5 in medium after 144 h of incubation at 30°C. Effect of inoculum ratio was also studied and found 3 ml/ 250 ml flask optimal. Effect of temperature on metabolic activity of the culture was quantified in terms of enthalpy, entropy and Gibbs energy demand for product formation and its inactivation. Activation enthalpy (MID.) of thermal inactivation for the mutant cells of (α - &β-galactosidase were lower (18.0 KJ/mol and 31.6 KJ/mol) than that of its parent (40.0 KJ/mol and 55.0 KJ/mol), which indicated that deactivation did not decrease faster with temperature and mutant had better stability than that of its parent. The activation entropy values of thermal inactivation by both cultures were very low (-234 and -260 J/mol/k) which suggested that inactivation phenomenon implied a little disorderness during the growth on substrate up to 40°C.β-Galactosidases from parent- and mutant-derived Aspergillus niger were purified by a combination of ammonium sulphate precipitation, anion exchange, hydrophobic interaction and gel filtration chromatography on Pharmacia FPLC unit. The five-step purification procedure from parent- and mutant-derived Aspergillus niger resulted into 9.4 and 15.6-fold purification with recovery of 16 and 15%, respectively. The purity of β-galactosidases was confirmed on 10% SDS-PAGE, which displayed a single band. The native and sub-unit molecular weights of both strains were 70 kDa and 67.6 kDa, respectively. The purified β-galactosidases were further characterized and found that both parent and mutant-derived β-galactosidase showed same pH (3.5-5.0 at 50 oC). The enzymes were stable at pH range of 2.6-8.2. The studies also revealed that both the enzymes have same optimum temperature (50 oC). The low value of Ea (24.1 kJ/mol) for the mutant-derived β-galactosidase indicated that it required less energy for hydrolysis of substrate and was more active at higher temperature than β-galactosidase (38.9 kJ/mol) of parent strain. The Vmax value of mutant-derived β-galactosidase (333 U/mg protein/m in) indicated 4.5 fold more activity than that of parent strain. The specificity constant (Vmax/Km) confirmed that β-galactosidase of mutant was more specific for pNPG as compared to parental because it was about 4-fold more specific than that of parent. It was investigated that enzymes involved two types of ionizable groups for hydrolysis of pNPG with pKal and pKa2. Due to mutation, conformation of active site of enzyme was slightly changed, the pKa2 of active site presented a significant shift from 6.25 to 6.9 as compared to parental-derived β-galactosidase. The Km values (0.118,0.333) provided evidence that pNPG had more affinity to the parental enzyme than the mutant one. The Gibbs free energy (ÎG*) for activation of unfolding of transition state of β-galactosidase from parent and mutant strains at 58 oC was same. Mutant-derived p-galactosidase showed higher values for free energy for transition state formation (ΔGE-T*) -18.87KJ/mole. Kinetic and thermodynamic studies revealed that mutant-derived enzyme exhibited longer half-life (t1/2) at 45, 48 and 51 oC as compared to parental-derived β-galactosidase, which supported that mutant-derived β-galactosidase is thermodynamically better. The mutant- derived β-galactosidase presented higher stability against urea and proteases (tl/2: 7.0 h) as compared to parental-derived p-galactosidase (t1/2: 22.6 h). These studies revealed that mutation imparted significantly better changes in the genetic make up of mutant derivative