CLONING OF 2 XYLANASE GENES FROM THE NEWLY ISOLATED ACTINOMYCETE ACTINOMADURA SP STRAIN FC7 AND CHARACTERIZATION OF THE GENE-PRODUCTS

A screening program was established to find actinomycetes that produce xylanases able to hydrolyze xylan chains in a hemicellulose liquor (a by-product of steam treatment of the lignocellulosic biomass) at moderately acidic pH (4.0) and high temperature (70 degrees C). The first step involved a selection for xylanolytic actinomycetes having optimal growth at 50-60 degrees C. In the second step, the crude enzymes produced by the selected actinomycetes were compared for their xylan hydrolysis rates at pH 5 and 60 degrees C versus pH 4 and 70 degrees C. The crude enzymes produced by strain FC7 retained 65% of their activity under the more stringent of the two conditions. FC7 was identified as a member of the genus Actinomadura by chemotaxonomic procedures. A gene bank of FC7 was constructed using the shuttle vector pFD666. The plasmids were introduced into a periplasmic-leaky Escherichia coli host to rapidly detect xylanolytic recombinants. Two classes of recombinants were isolated. The plasmids pJF1 and pJF6 were introduced into a xylanase- and cellulase-negative mutant of Streptomyces lividans, allowing overproduction and subsequent purification of two different xylanases, Xyl I and Xyl II. Both enzymes were classified in the low isoelectric point group of xylanases. In the presence of 100 mu g/mL of bovine serum albumin, the half-life of Xyl I at pH 4 and 70 degrees C was 22 h.