Cloning of a Family 11 Xylanase Gene from Bacillus amyloliquefaciens CH51 Isolated from Cheonggukjang

Abstract

Bacillus amyloliquefaciens CH51, an isolate from cheonggukjang, Korean fermented soyfood, secretes several enzymes into culture medium. A gene encoding 19 kDa xylanase was cloned by PCR. Sequencing showed that the gene encoded a glycohydrolase family 11 xylanase and named xynA. xynAHis, xynA with additional codons for his-tag, was overexpressed in Escherichia coli BL21(DE3) using pET-26b(+). XynAHis was purified using HisTrap affinity column. Km and Vmax of XynAHis were 0.363 mg/ml and 701.1 μmol/min/mg, respectively with birchwood xylan as a substrate. The optimum pH and temperature were pH 4 and 25 °C, respectively. When xynA was introduced into Bacillus subtilis WB600, active XynA was secreted into culture medium.

Keywords: Bacillus amyloliquefaciens; Family 11 xylanase; Overexpression; Purification.

Fig. 1

SDS-PAGE and zymogram of purified XynAHis. M, Dok-Do-MARK proteins (Elpisbio, Daejeon, Korea); 1, coomassie blue stained XynAHis; 2, zymogram. XynAHis was purified by HisTrap affinity column and 5 μg was analyzed on 15 % acrlyamide gel

Fig. 2

Optimum pH (a) and pH stability (b) of XynAHis. a Purified XynAHis (10 μg) was incubated in different pHs for 30 min at 37 °C b. XynAHis was incubated in different pHs for 3 h. Then the remaining activity was measured in each case

Fig. 3

Optimum temperature (a) and temperature stability (b) of XynAHis. a XynAHis (1 μg) in 0.1 M citrate buffer (pH 4.0) was incubated for 30 min at different temperatures. Then the activity was measured. b XynAHis was incubated for 1 h at different temperatures. Sample was taken every 10 min and the activity was measured. Filled circle, 37 °C; filled square, 45 °C; filled triangle, 50 °C

Fig. 4

SDS-PAGE and zymogram of B. subtilis strains. M, Dok-Do-MARK proteins (Elpisbio); 1, culture supernatant from B. subtilis WB600 [pHY300PLK]; 2, culture supernatant from B. subtilis WB600 [pHxyna]; 3, concentrated culture supernatant from B. subtilis WB600 [pHY300PLK]; 4, concentrated culture supernatant from B. subtilis WB600 [pHxyna]. Culture supernatant was concentrated by ammonium sulfate precipitation (80 %). Each 10 μg sample was analyzed on 15 % acrlyamide gel