A gene encoding a novel extremely thermostable 1,4-β-xylanase isolated directly from an environmental DNA sample

Anwar Sunna, Peter L. Bergquist

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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°C and pH 6.0, and was extremely thermostable at 90°C. The enzyme showed high specificity toward different xylans and xylooligosaccharides.

LanguageEnglish
Pages63-70
Number of pages8
JournalExtremophiles
Volume7
Issue number1
DOIs
Publication statusPublished - 2003

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Endo-1,4-beta Xylanases
Ribosomal DNA
Glycoside Hydrolases
Polymerase Chain Reaction
Sequence Analysis
DNA
Genes
Xylans
Archaea
Enzymes
rRNA Genes
Restriction Fragment Length Polymorphisms
Open Reading Frames
Walking
Escherichia coli
Bacteria
Proteins

Cite this

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title = "A gene encoding a novel extremely thermostable 1,4-β-xylanase isolated directly from an environmental DNA sample",
abstract = "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°C and pH 6.0, and was extremely thermostable at 90°C. The enzyme showed high specificity toward different xylans and xylooligosaccharides.",
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A gene encoding a novel extremely thermostable 1,4-β-xylanase isolated directly from an environmental DNA sample. / Sunna, Anwar; Bergquist, Peter L.

In: Extremophiles, Vol. 7, No. 1, 2003, p. 63-70.

Research output: Contribution to journalArticleResearchpeer-review

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