A gene encoding a novel extremely thermostable 1,4-beta-xylanase isolated directly from an environmental DNA sample.
Small-subunit (SSU) rRNA genes (rDNA) were amplified by PCR from a hot pool environmental DNA sample using Bacteria- or Archaea-specific rDNA primers. Unique rDNA types were identified by restriction fragment length polymorphism (RFLP) analysis and representative sequences were determined. Family 10 glycoside hydrolase consensus PCR primers were used to explore the occurrence and diversity of xylanase genes in the hot pool environmental DNA sample. Partial sequences for three different xylanases were obtained and genomic walking PCR (GWPCR), in combination with nested primer pairs, was used to obtained a unique 1,741-bp nucleotide sequence. Analysis of this sequence identified a putative XynA protein encoded by the xynA open reading frame. The single module novel xylanase shared sequence similarity to the family 10 glycoside hydrolases. The purified recombinant enzyme, XynA expressed in E. coli exhibited optimum activity at 100 degrees C and pH 6.0, and was extremely thermostable at 90 degrees C. The enzyme showed high specificity toward different xylans and xylooligosaccharides.
Sunna
Anwar
A
Department of Biological Sciences, Macquarie University, Sydney, Australia. asunna@rz.uni-potsdam.de
Bergquist
Peter L
PL
D000595:Amino Acid Sequence; D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics* / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D043364:Endo-1,4-beta Xylanases; D004795:Enzyme Stability; D019766:Genes, Archaeal; D005798:Genes, Bacterial; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009520:New Zealand; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014871:Water Microbiology; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D019641:DNA, Archaeal; D004269:DNA, Bacterial; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases