CRYSTALLIZATION AND PRELIMINARY-X-RAY ANALYSIS OF THE THERMOSTABLE ALKALINE-TOLERANT XYLANASE FROM BACILLUS-STEAROTHERMOPHILUS T-6

The extracellular thermostable xylanase (XT-6) produced by the thermop hilic bacterium Bacillus stearothermophilus T-6 was shown to bleach pu lp optimally at pH9 and 338K, and was successfully used in a large-sca le biobleaching mill trial. The xylanase gene was cloned and sequenced . The mature enzyme consists of 379 amino acids with a calculated mole cular weight of 43 808 and pl of 9.0. Crystallographic studies of XT-6 were initiated to study the mechanism of catalysis as well as to prov ide a structural basis for rational introduction of enhanced thermosta bility by site-specific mutagenesis. This report describes the crystal lization and preliminary crystallographic characterization of the nati ve XT-6 enzyme. The most suitable crystals were obtained by the vapor- diffusion method using ammonium sulfate and 2-methyl-2,4-pentanediol a s an organic additive. The crystals belong to a primitive trigonal cry stal system (space group P3(1) or P3(2)) with room-temperature cell di mensions of a = b = 114.9 and c = 122.6 Angstrom. At 103 K the volume of the unit cell decreased significantly with observed dimensions of a = b = 112.2 and c = 122.9 Angstrom. These crystals are mechanically s trong and diffract X-rays to better than 2.2 Angstrom resolution. The crystals exhibit considerable radiation damage at room temperature eve n at relatively short exposures to X-rays. A full 2.3 Angstrom resolut ion diffraction data set (99.8% completeness) has recently been collec ted on flash-frozen crystals at 103 K using synchrotron radiation. Two derivatives of XT-6 were recently prepared. In the first derivative, a unique Cys residue replaced Glu265, the putative nucleophile in the active site. The second derivative was selenomethionyl xylanase which was produced biosynthetically. These derivatives have been crystallize d and the resulting crystals were shown to be isomorphous to the nativ e crystals and diffract X-rays to comparable resolutions.