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

• Published in: Applied microbiology and biotechnology 2012-02, Vol.93 (4), p.1503-1512
• Main Authors: Wang, Shu-Yun, Hu, Wei, Lin, Xiao-Yu, Wu, Zhi-Hong, Li, Yue-Zhong
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Analysis; Bacteria; Base Composition; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Catalytic Domain; Chromatography, Affinity; Cloning; Cloning, Molecular; Cold; Cold Temperature; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; E coli; Enzyme Stability; Enzymes; Escherichia coli; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Life Sciences; Microbial Genetics and Genomics; Microbiology; Molecular Sequence Data; Myxococcales - enzymology; Myxococcales - genetics; Peptides; Plasmids; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Saccharides; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sorangium cellulosum; Studies; Temperature; Xylans - metabolism; Xylosidases - genetics; Xylosidases - metabolism
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-011-3480-3

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 .