Xyn10A, a thermostable endoxylanase from Acidothermus cellulolyticus 11B

Abstract

We cloned and purified the major family 10 xylanase (Xyn10A) from Acidothermus cellulolyticus 11B. Xyn10A was active on oat spelt and birchwood xylans between 60°C and 100°C and between pH 4 and pH 8. The optimal activity was at 90°C and pH 6; specific activity and K(m) for oat spelt xylan were 350 μmol xylose produced min⁻¹ mg of protein⁻¹ and 0.53 mg ml⁻¹, respectively. Based on xylan cleavage patterns, Xyn10A is an endoxylanase, and its half-life at 90°C was approximately 1.5 h in the presence of xylan.

FIG. 1.

Expression of xyn10A in A. cellulolyticus at mid-exponential (open bars) and stationary (filled bars) growth phases. Densitometry-based relative intensity values of reverse transcriptase PCR (RT-PCR) products are plotted along the y axis. RNA was extracted using the RNeasy plant minikit (Qiagen). Primers specific to the xyn10A gene (5′-CAAAGGAAAGATCTGGCAATG-3′ and 5′-TGAGCATCCCGTCGTAGTAGT-3′) were used to amplify a 485-bp product using the Qiagen OneStep RT-PCR kit. Expression of the housekeeping gyrB (Acel_0005) gene was used to normalize for the RNA (data not shown). Identical results were obtained in two independent experiments; data from one experiment are presented.

FIG. 2.

Heterologous expression and purification of recombinant Xyn10A. (A) Zymogram assay showing xylanase activity following electrophoresis of crude cell extracts. Lanes: ST, molecular weight standards; 1 and 3, crude cell extracts from DH5α(pK19) (vector control); 2 and 4, crude cell extracts from DH5α(pK19-xyn10A). Samples in lanes 3 and 4 were heated at 65°C for 15 min prior to loading. (B) SDS-PAGE (10% gel) showing purification of Xyn10A. Lanes: A, molecular weight standards; B, crude cell extract from DH5α(pK19-xyn10A); C, heat-treated extract from DH5α(pK19-xyn10A); D, concentrated fractions from the hydroxyapatite column.

FIG. 3.

Time course of Xyn10A product formation with birchwood (A) and oat spelt (B) xylans, using TLC. Xylan substrates (2% in 10 mM phosphate buffer, pH 6) were incubated with purified Xyn10A at 90°C. Lanes 1 and 8, standards: xylotetraose (X4) and xylotriose (X3) (Megazyme, Wicklow, Ireland) and xylose (X1). Lane 2, unreacted xylan; lanes 3 to 7, xylan with increased incubation times in the presence of purified Xyn10A (10, 20, 40, 60, and 240 min, respectively).

FIG. 4.

Stabilization of purified Xyn10A by xylans. (A) Purified Xyn10A was diluted 1:20 with 4% oat spelt xylan (white), 4% birchwood xylan (gray), or phosphate buffer (black) and incubated at 90°C for the times indicated. Rates of reducing sugar formation were determined and were linear for all samples. Activities relative to unheated Xyn10A (dark gray) are reported. Results are averages of three independent experiments; error bars indicate standard deviations. (B) Long-term thermostability of Xyn10A at 90°C in the presence of 4% oat spelt xylan. Experiments were performed as described for panel A.