Optimization and partial purification of beta-galactosidase production by Aspergillus niger isolated from Brazilian soils using soybean residue

Abstract

β-Galactosidases are widely used for industrial applications. These enzymes could be used in reactions of lactose hydrolysis and transgalactosylation. The objective of this study was the production, purification, and characterization of an extracellular β-galactosidase from a filamentous fungus, Aspergillus niger. The enzyme production was optimized by a factorial design. Maximal β-galactosidase activity (24.64 U/mL) was found in the system containing 2% of a soybean residue (w/v) at initial pH 7.0, 28 °C, 120 rpm in 7 days. ANOVA of the optimization study indicated that the response data on temperature and pH were significant (p < 0.05). The regression equation indicated that the R² is 0.973. Ultrafiltration at a 100 and 30 kDa cutoff followed by gel filtration and anion exchange chromatography were carried out to purify the fungal β-galactosidase. SDS-PAGE revealed a protein with molecular weight of approximately 76 kDa. The partially purified enzyme showed an optimum temperature of 50 °C and optimum pH of 5.0, being stable under these conditions for 15 h. The enzyme was exposed to conditions approaching gastric pH and in pepsin's presence, 80% of activity was preserved after 2 h. These results reveal a A. niger β-galactosidase obtained from residue with favorable characteristics for food industries.

Keywords: Agroindustrial residues; Fungi; Optimization; Purification; β-Galactosidase.

Fig. 1

β-galactosidase activity in different soybean products, after 7 days of submerse fermentation at 120 rpm and 28 °C

Fig. 2

Response surface curve of β-galactosidase activity affected by temperature and pH using the 2-factor central composite design

Fig. 3

Time courses of β-galactosidase production by Aspergillus niger under optimized conditions (120 rpm, 28 °C, initial medium pH 7), using medium supplemented with 2% of soybean residue. Medium pH (∙∙∙∙), protein (mg/mL) (---) and β-galactosidase activity (—)

Fig. 4

a SDS-PAGE of partially purified β-galactosidase. Lane 1: Molecular mass markers, mass indicated alongside. Lane 2: S-200 peak. Lane3: DEAE peak. b β-galactosidase activity zymogram from DEAE peak

Fig. 5

a Activity versus pH profiles of the partially purified β-galactosidase. Enzyme activity is plotted as a % value relative to the activity displayed at the enzyme’s optimum pH. Error bars indicate the standard deviation of the measured data values from the mean, n = 3. b Activity versus temperature profiles of the partially purified β-galactosidase. Enzyme activity is plotted as a % value relative to the activity displayed at the enzyme’s optimum temperature. Error bars indicate the standard deviation of the measured data values from the mean, n = 3. c Temperature stability of the β-galactosidase partially purified from A. niger at 4 °C, 50 °C and 70 °C

Fig. 6

The effect of in vitro simulated fasted gastric (SGF) conditions on partially purified β-galactosidase. Control: enzyme incubated with buffer pH 2.0. Enzyme + SGF (NaCl 2%, bovine pepsin 0.32% and HCl 7%). *Indicates statistical difference in the ANOVA Tukey test with significance level (p < 0.05) between control and enzyme + SGF