Studies on an alkali-thermostable xylanase from Aspergillus fumigatus MA28

Abstract

An alkalitolerant fungus, Aspergillus fumigatus strain MA28 produced significant amounts of cellulase-free xylanase when grown on a variety of agro-wastes. Wheat bran as the sole carbon source supported higher xylanase production (8,450 U/L) than xylan (7,500 U/L). Soybean meal was observed to be the best nitrogen source for xylanase production (9,000 U/L). Optimum medium pH for xylanase production was 8 (9,800 U/L), though, significant quantities of the enzyme was also produced at pH 7 (8,500 U/L), 9 (8,200 U/L) and 10 (4,600 U/L). The xylanase was purified by ammonium sulphate precipitation and carboxymethyl cellulose chromatography, and was found to have a molecular weight of 14.4 kDa with a V(max) of 980 μmol/min/mg of protein and a K(m) of approximately 4.9 mg/mL. The optimum temperature and pH for enzyme activity was 50 °C and pH 8, respectively. However, the enzyme also showed substantial residual activity at 60-70 °C (53-75%) and at alkaline pH 8-9 (56-88%).

Fig. 1

Growth and time-course of xylanase production by Aspergillus fumigatus MA28. The organism was cultivated under shaking (180 rpm) at 30 °C. Amount of biomass was determined on dry weight basis (in hot air oven at 90 °C), and xylanase activity was assayed by determining the ability of enzyme to cause release of reducing sugars from xylan

Fig. 2

Xylanase production from various agriculture-based carbon sources. Fermentation was executed under shaking (180 rpm) at 30 °C. Black bar indicates xylanase production when xylan was used as carbon source (5 g/L), while the colourless bars show xylanase production when xylan was replaced with either of the agriculture-based carbon sources (10 g/L)

Fig. 3

Xylanase production by Aspergillus fumigatus MA28 using various nitrogen sources. Fermentation was executed under shaking (180 rpm) at 30 °C. Black bar indicates xylanase production when ammonium salts (ammonium sulphate and ammonium acetate) were used as nitrogen source while the colourless bars show when ammonium salts were replaced with various nitrogen sources (5 g/L)

Fig. 4

Effect of initial pH of medium on xylanase production by A. fumigatus MA28. The initial pH of the production medium was adjusted at pH 5–10 using either HCl (0.1 M) or NaOH (0.1 M), and fermentation was carried out under shaking (180 rpm) at 30 °C

Fig. 5

Specific activity of xylanase in different fractions eluted from carboxymethyl cellulose chromatography column. Elution was done by using increasing concentration of NaCl (0.25–1 M) solution

Fig. 6

Effect of temperature on activity of A. fumigatus MA28 xylanase. Activity assay was conducted at different temperatures (30–100 °C) by incubating the enzyme assay mixture in water bath set at appropriate temperature

Fig. 7

Effect of pH on activity of A. fumigatus MA28 xylanase. The activity assay was conducted at different pH (3–10) using appropriate buffers. The substrate solution used in the enzyme assay mixture was prepared either in citrate buffer (pH 3, 4, 5 and 6), tris buffer (pH 7, 8 and 9) or glycine-NaOH (pH 10) to expose the enzyme to different pH

Fig. 8

Thermostability of A. fumigatus MA28 xylanase. The enzyme (without substrate) was pre-incubated at different temperatures (50–100 °C) for varying time periods (30 and 60 min) and then assayed for residual activity. Initial activity was considered as 100%

Fig. 9

Stability of A. fumigatus MA28 xylanase at different pH. The enzyme (without substrate) was pre-incubated for 30 and 60 min at different pH using appropriate buffers: citrate buffer (pH 6), tris buffer (pH 7, 8 and 9) and glycine-NaOH (pH 10), and then assayed for residual activity. Initial activity was considered as 100%

Fig. 10

Effect of various additives and cations on xylanase activity. Black bar indicates activity without any additive/ion (control) while the colourless bars show xylanase activity when either of the additive or ion was included in enzyme assay mixture at final concentration of 10 mM