Optimization of β-1,4-Endoxylanase Production by an Aspergillus niger Strain Growing on Wheat Straw and Application in Xylooligosaccharides Production

Abstract

Plant biomass constitutes the main source of renewable carbon on the planet. Its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on Earth. The main enzymes involved in plant biomass degradation are glycosyl hydrolases, and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was used for hemicellulase production under solid-state fermentation using wheat straw as single-carbon source. Physicochemical parameters for the production of an endoxylanase were optimized by using a One-Factor-at-a-Time (OFAT) approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold, and 1.41- fold purification was achieved after ultrafiltration and ion-exchange chromatography, with about 6.2% yield. The highest activity of the purified xylanase was observed at 50 °C and pH 6. The enzyme displayed high thermal and pH stability, with more than 90% residual activity between pH 3.0-9.0 and between 30-40 °C, after 24 h of incubation, with half-lives of 30 min at 50 and 60 °C. The enzyme was mostly active against wheat arabinoxylan, and its kinetic parameters were analyzed (K_m = 26.06 mg·mL^-1 and V_max = 5.647 U·mg^-1). Wheat straw xylan hydrolysis with the purified β-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.

Keywords: enzymes; fungi; hemicellulose; hydrolysis; wastes; xylan.

Figure 1

Effect of incubation period (a), temperature (b), moisture agent level (c) and initial pH value (d) on the endoxylanase production of A. niger BG strain growing under SSF conditions.

Figure 2

Actual value vs. predicted value split from RSM design.

Figure 3

Interaction plots (a,c) and response surface plot (b,d) for the model of the intersection between moisture level (X₂) (a,b) vs. incubation time (X₄) or pH (X₃) (c,d) vs. incubation time (X₄) for endoxylanase production by A. niger BG strain.

Figure 4

SDS-PAGE (a) and zymogram analyses (b) of purified A. niger BG endoxylanase. (a) Lane 1, protein standards; lane 2, crude extract; lane 3, proteins after HiTrap QFF chromatography; lane 4, purified enzyme after Mono-Q column.

Figure 5

Effect of temperature, pH and different substrates on the purified xylanase activity produced by A. niger BG strain. (a) Optimum temperature (line) and temperature stability (bars). For optimal temperature, xylanase activity was assayed for 5 min with 1.8% xylan (as described in Section 3.2) but at temperatures between 30–90 °C. For thermal stability analysis, enzyme was incubated at the different temperatures and samples were taken after 0.5, 1, 2, 3, 4, 5, 6, and 24 h for measuring residual activity under standard conditions. (b) Optimum pH (line) and pH stability (bars). For optimum pH determination, the initial pH of the reaction was adjusted using different buffers: 50 mM sodium citrate buffer (pH 2.0–5.0), 50 mM sodium phosphate buffer (pH 6.0–8.0) and Tris HCl buffer (pH 9.0–10.0), and activity was measured for 5 min at 50 °C. For pH stability assays, the enzyme was incubated at different pH (using the same buffers) for the indicated time and activity was checked under standard conditions. (c) Xylanase specificity towards diverse substrates: the activity was measured at standard assay conditions and the relative activity was calculated for different substrates, considering beech wood xylan activity as 100%. WARX: wheat arabinoxylan.

Figure 6

TLC analysis of β-1,4-endoxylanase hydrolysed products from wheat straw xylan. Lanes: M: Markers (X1; xylose, X2; xylobiose, X3; xylotriose, X4; xylotetraose and X5; xylopentaose). 1: Negative control; 2–9: reaction products after 2–5 min, 3–15 min, 4–30 min, 5–1 h, 6–2 h, 7–4 h, 8–6 h, and 9–24 h.