ORIGINAL PAPER Anwar Sunna Æ Peter L. Bergquist A gene encoding a novel extremely thermostable 1,4-b-xylanase isolated directly from an environmental DNA sample Received: 1 February 2002 / Accepted: 28 August 2002 / Published online: 15 October 2002 Ó Springer-Verlag 2002 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 restric- tion 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 ob- tained and genomic walking PCR (GWPCR), in com- bination 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 dif- ferent xylans and xylooligosaccharides. Keywords 16S rDNA Æ b-xylanase Æ Environmental DNA Æ Genomic walking PCR (GWPCR) Æ Thermostability Introduction Xylans are heterogeneous polysaccharides consisting of a main chain of 1,4-linked b-D-xylopyranosyl residues that often carry acetyl, arabinosyl, and glucuronosyl substituents. The action of the main xylanolytic enzyme, b-endoxylanase (1,4-b-D-xylan xylanohydrolase, EC 3.2.1.8) is to convert polymeric xylan to xylooligosac- charides (Biely 1985). Xylanases are classified into two families, 10 and 11, according to the similarity of amino- acid sequences of their catalytic domain in hydrophobic cluster analyses (Henrissat 1991). The classical approach to obtaining new xylanases has been based on the screening and isolation of xy- lanolytic microorganisms. However, typically only a small fraction (<1%) of naturally occurring microor- ganisms can be cultivated using standard techniques (Amann et al. 1995). Several approaches have been de- veloped recently to overcome this limitation. One is based on the use of environmental DNA for the con- struction of DNA libraries and direct screening for functional gene products (Cottrell et al. 1999; Henne et al. 2000). An alternative method is to use PCR primers based on conserved regions of known genes to amplify new genes from DNA extracted from noncultured bio- mass (Seow et al. 1997; Cottrell et al. 2000). In this study we used a modified genomic walking PCR (GWPCR) technique to retrieve xylanase genes from a hot pool environmental DNA sample. We report here the clon- ing, sequencing, and expression of a novel xylanase, XynA, along with some biochemical properties of the purified enzyme. Materials and methods Bacterial strains and genomic DNA Escherichia coli INVaF¢ One-Shot cells (Invitrogen, Carlsbad, CA, USA) were used as the bacterial host for all DNA cloning and expression studies. Genomic DNA was prepared as described previously (Morris et al. 1995) from environmental samples from Extremophiles (2003) 7:63–70 DOI 10.1007/s00792-002-0296-1 Communicated by G. Antranikian A. Sunna (&) Æ P. L. Bergquist Department of Biological Sciences, Macquarie University, Sydney, Australia P. L. Bergquist Division of Molecular Medicine, University of Auckland Medical School, Auckland, New Zealand Present address: A. Sunna Physikalische Biochemie, Universita¨t Potsdam, Karl-Liebknecht-Strasse 24-25, Haus 25, 14476 Golm, Germany E-mail: asunna@rz.uni-potsdam.de Tel.: +49-331-9775245 Fax: +49-331-9775062