Cloning of two xylanase genes from the newly isolated actinomycete Actinomadura sp. strain FC7 and characterization of the gene products

Abstract

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 °C). The first step involved a selection for xylanolytic actinomycetes having optimal growth at 50–60 °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 °C versus pH 4 and 70 °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 pJFl 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 μg/mL of bovine serum albumin, the half-life of Xyl I at pH 4 and 70 °C was 22 h.Key words: xylanase, thermophilic actinomycetes, Actinomadura, thermostability.