EVIDENCE FOR A GENERAL ROLE FOR NONCATALYTIC THERMOSTABILIZING DOMAINS IN XYLANASES FROM THERMOPHILIC BACTERIA

A genomic library of Clostridium thermocellum DNA constructed in lambd a ZAPII was screened for xylanase-expressing clones. Cross-hybridizati on experiments revealed a new xylanase gene isolated from the gene lib rary, which was designated xynY. The encoded enzyme, xylanase Y (XYLY) , displayed features characteristic of an endo-beta 1,4-xylanase: the enzyme rapidly hydrolysed oat spelt, wheat and rye arabinoxylans and w as active against methyl-umbelliferyl-beta-D-cellobioside, but did not hydrolyse any cellulosic substrates. The pH and temperature optima of the enzyme were 6.8 and 75 degrees C respectively, and the recombinan t XYLY, expressed by Escherichia coli had a maximum M(r) of 116 000. T he nucleotide sequence of xynY contained an open reading frame of 3228 bp encoding a protein of predicted M(r) 120 105. The encoded enzyme c ontained a typical N-terminal 26-residue signal peptide, followed by a 164 amino acid sequence, designated domain A, that was not essential for catalytic activity. Downstream of domain A was a 351-residue xylan ase Family F catalytic domain, followed by a 180-residue sequence that exhibited 28 % sequence identity with a thermostable domain of Thermo anaerobacterium saccharolyticum xylanase A. The C-terminal portion of XYLY comprised the U-residue duplicated docking sequence found in all other C. thermocellum plant cell wall hydrolases that are constituents of the bacterium's multienzyme complex, termed the cellulosome, follo wed by a 286-residue domain which exhibited 32 % sequence identity wit h the N-terminal region of C. thermocellum xylanase Z. The enzyme did not contain linker sequences found in other C. thermocellum plant cell wall hydrolases. Analysis of truncated forms of XYLY and hybrid prote ins, comprising segments of XYLY fused to the E. coli maltose binding domain, confirmed that XYLY contained a central catalytic domain and a n adjacent thermostable domain. The C-terminal domain did not bind to cellulose or xylan. Western blot analysis using antiserum raised again st XYLY showed that the xylanase was located in the cellulosome and di d not appear to be extensively glycosylated. The non-catalytic domains of XYLY are discussed in relation to the general stability of thermop hilic xylanases.