High levels of β-xylosidase in Thermomyces lanuginosus: potential use for saccharification

Abstract

A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and its β-xylosidases optimization in response to agro-industrial residues was performed. Using statistical approach as a strategy for optimization, the induction of β-xylosidases activity was evaluated in residual corn straw, and improved so that the optimum condition achieved high β-xylosidases activities 1003U/mL. According our known, this study is the first to show so high levels of β-xylosidases activities induction. In addition, the application of an experimental design with this microorganism to induce β-xylosidases has not been reported until the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60°C showing better thermostability at 55°C. The saccharification ability of β-xylosidase in the presence of hemicellulose obtained from corn straw raw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50°C. Our data strongly indicated that the β-xylosidases activities was not subjected to the effects of potential enzyme inhibitors often produced during fermentation process. These data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production.

Keywords: Atlantic Forest; Experimental design; Saccharification; Thermomyces lanuginosus; β-Xylosidase.

Fig. 1

(A) β-xylosidases activities in the crude extract of Thermomyces lanuginosus after 7 days of cultivation in mineral medium supplemented with 1% of various agro-industrial residues as a carbon source at 42 °C and at stationary conditions. (B) Production of the intracellular β-xylosidases enzyme of T. lanuginosus during 7 days in culture containing mineral medium supplemented with 1% of agro-industrial residues as a carbon source at 42 °C and agitation at 150 rpm. (C) Evaluation of fungal growth on different carbon sources and with different cultivation conditions.

Fig. 2

Response surfaces generated by analysis of variance for the production of β-xylosidases in terms of (A) temperature and residual corn straw, (B) yeast extract and corn straw and (C) yeast extract and corn straw. The darkest portion of the graph shows higher levels of enzyme activity.

Fig. 3

Desirability for the production of T. lanuginosus β-xylosidase.

Fig. 4

(A) Effect of pH on the activity of intracellular β-xylosidase of T. lanuginosus incubated in McIlvaine buffer (pH 5.0–7.5) followed by determination of β-xylosidase using ρNPX as a substrate. (B) Effect of temperature on the activity of intracellular β-xylosidase of T. lanuginosus incubated in McIlvaine buffer (pH 5.5) during 10 min at temperatures of 50–70 °C with subsequent standard determination of β-xylosidase.

Fig. 5

Thermostability profile of the intracellular T. lanuginosus β-xylosidase activity. (A) Thermostability displayed in the top three temperatures of 55 °C, 60 °C and 65 °C during 210 min of incubation time. (B) The enzyme stability at pH 5.5 at temperatures of 55 °C, 60 °C and 65 °C during 150 min.

Fig. 6

Performance T. lanuginosus β-xylosidases activities during saccharification. Relative activity after hydrolysis by intracellular β-xylosidase of T. lanuginosus for 12 h in xylan from beechwood (square) (1%, w/v) and hemicellulose from corn straw (circle) (1%, w/v) at 37 °C (closed circle and closed square) and 50 °C (open circle and open square). Relative activity exhibited by T. lanuginosus β-xylosidase in the absence and presence of 200 mM xylose after 12 h at 37 °C and 50 °C.

Fig. 7

Analysis of thin layer chromatography. TLC of the hydrolysis of T. lanuginosus β-xylosidase products obtained after 12 h of incubation with xylan from beechwood (1%, w/v) and hemicellulose from corn straw (1%, w/v) at 37 °C (A) and 50 °C (B). P, xylose, xylobiose and xylotriose; XL, xylan from beechwood (1%, w/v); H, hemicellulose from corn straw (1%, w/v); XLC, xylan from beechwood (1%, w/v) control; HC, hemicellulose from corn straw (1%, w/v) control.