Purification and biochemical characterization of an extracellular fructosyltransferase enzyme from Aspergillus niger sp. XOBP48: implication in fructooligosaccharide production

Abstract

An extracellular fructosyltransferase (Ftase) enzyme with a molar mass of ≈70 kDa from a newly isolated indigenous coprophilous fungus Aspergillus niger sp. XOBP48 is purified to homogeneity and characterized in this study. The enzyme was purified to 4.66-fold with a total yield of 15.53% and specific activity of 1219.17 U mg^-1 of protein after a three-step procedure involving (NH₄)₂SO₄ fractionation, dialysis and anion exchange chromatography. Ftase showed optimum activity at pH 6.0 and temperature 50 °C. Ftase exhibited over 80% residual activity at pH range of 4.0-10.0 and ≈90% residual activity at temperature range of 40-60 °C for 6 h. Metal ion inhibitors Hg²⁺ and Ag⁺ significantly inhibited Ftase activity at 1 mmol concentration. Ftase showed K _m, v _max and k _cat values of 79.51 mmol, 45.04 µmol min^-1 and 31.5 min^-1, respectively, with a catalytic efficiency (k _cat/K _m) of 396 µmol^-1 min^-1 for the substrate sucrose. HPLC-RI experiments identified the end products of fructosyltransferase activity as monomeric glucose, 1-kestose (GF₂), and 1,1-kestotetraose (GF₃). This study evaluates the feasibility of using this purified extracellular Ftase for the enzymatic synthesis of biofunctional fructooligosaccharides.

Keywords: 1,1-Kestotetraose; 1-Kestose; Aspergillus niger; Fructooligosaccharides; Fructosyltransferase; Zymography.

Fig. 1

SDS-PAGE of purified Ftase. a A 12% SDS-PAGE showing: Pre-stained protein marker (Lane M), ≈200 µg total protein from crude enzyme preparation (Lane 1), ≈200 µg total protein from dissolved precipitates from 70% (NH₄)₂SO₄ saturation (Lane 2), ≈200 µg total protein from dialyzed fraction of 70% (NH₄)₂SO₄ saturation (Lane 3); b Pre-stained protein marker (Lane M), ≈200 µg total protein from concentrated pooled fractions from HiTrap QFF anion exchange chromatography (Lane 1)

Fig. 2

Zymography and TTC activity assay. a Zymogram showing the in-gel activity of Ftase (Lane 1) and Molecular weight marker (Lane M); b TTC activity assay showing the appearance of red zone indicating FOS production during purified Ftase and sucrose reaction

Fig. 3

Optimum pH and pH stability of purified Ftase. a Ftase activity in U mL⁻¹ determined by performing the enzyme assays at pH 2–12. ≈100 µg total protein from pooled fractions was incubated with 5% (w/v) sucrose dissolved in different pH buffers in a total 3 mL reaction mixture and incubated as described in “Fructosyltransferase (Ftase) assay”. b the stability of Ftase at pH 2–12. ≈100 µg total protein from pooled fractions was incubated in pH 2–12 buffers for 6 h and the assays were performed by adding 5% (w/v) sucrose dissolved in optimum pH buffer and activity was measured at optimum pH and temperature as described above. The % residual activity was calculated as compared to the activity shown at optimum conditions. The error bars indicate the standard deviations of three replicates

Fig. 4

Optimum temperature and temperature stability of purified Ftase. a Ftase activity in U mL⁻¹ determined by conducting the enzyme assays at temperature from 30 to 80 °C. ≈100 µg total protein from pooled fractions was incubated with 5% (w/v) sucrose dissolved in pH 6.5 buffer in a total 3 mL reaction mixture and incubated at temperature from 30 to 80 °C as described in “Fructosyltransferase (Ftase) assay”. b the stability of Ftase at temperature 40–80 °C at 10 °C interval. ≈200 µg total protein from pooled fractions was incubated at temperature 40–80 °C at 10 °C interval for 6 h in pH 6.5 buffer and cooled to room temperature after incubation period. The reaction was commenced by adding 5% (w/v) sucrose dissolved in optimum pH 6.5 buffer and optimum temperature 50 °C as described above. The % residual activity was calculated as compared to the activity shown at optimum conditions. The error bars indicate the standard deviations of three replicates

Fig. 5

TLC and HPLC-RI analysis of the Ftase and sucrose reaction products. a TLC profile showing standards of fructose (1), glucose (2), sucrose (3), 1- kestose (4), nystose (5), fructofuranosyl nystose (6) and reaction products (7); b The products of the reaction between of purified Ftase and sucrose analysed by HPLC-RI. The retention time and peak area of the FOS and glucose liberated were compared to the retention time and peak area of standards for the purpose of quantification