title: 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.
authors.affiliation: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, China.
authors.forename: Shu-Yun
authors.initials: SY
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Hu
authors.forename: Xiao-Yu
authors.initials: XY
authors.lastname: Lin
authors.forename: Zhi-Hong
authors.initials: ZH
authors.lastname: Wu
authors.forename: Yue-Zhong
authors.initials: YZ
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001482:Base Composition; D020134:Catalytic Domain; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003080:Cold Temperature; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009231:Myxococcales / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D014990:Xylans; D014995:Xylosidases