A novel cold-active xylanase from the cellulolytic myxobacterium Sorangium cellulosum So9733-1: gene cloning, expression, and enzymatic characterization.

ABSTRACT

The cellulolytic myxobacterium Sorangium cellulosum is able to efficiently degrade many kinds of polysaccharides, but none of the enzymes involved have been characterized. In this paper, a xylanase gene (xynA) was cloned from S. cellulosum So9733-1 using thermal asymmetric interlaced PCR. The gene is composed of 1,209 bp and has only 52.27% G + C content, which is much lower than that of most myxobacterial DNA reported (67-72%). Gene xynA encodes a 402 amino acid protein that contains a single catalytic domain belonging to the glycoside hydrolase family 10. The novel xylanase gene, xynA, was expressed in Escherichia coli BL21 (DE3) and the recombinant protein (r-XynA) was purified by Ni-affinity chromatography. The r-XynA had the optimum temperature of 30-35°C and exhibited 33.3% activity at 5°C and 13.7% activity at 0°C. Approximately 80% activity was lost after 20-min pre-incubation at 50°C. These results indicate that r-XynA is a cold-active xylanase with low thermostability. At 30°C, the K (m) values of r-XynA on beechwood xylan, birchwood xylan, and oat spelt xylan were 25.77 ± 4.16, 26.52 ± 4.78, and 38.13 ± 5.35 mg/mL, respectively. The purified r-XynA displayed optimum activity at pH 7.0. The activity of r-XynA was enhanced by the presence of Ca(2+). The r-XynA hydrolyzed beechwood xylan, birchwood xylan, and xylooligosaccharides (xylotriose, xylotetraose, and xylopentose) to produce primarily xylose and xylobiose. To our knowledge, this is the first report on the characterization of a xylanase from S. cellulosum.