A single domain thermophilic xylanase can bind insoluble xylan: evidence for surface aromatic clusters

A clone expressing xylanase activity in Escherichia colt has been selected from a genomic plasmid library of the thermophilic Bacillus strain D3. Subc loning from the 9-kb insert located the xylanase activity to a 2.7-kb HindI I/BamHI fragment. The DNA sequence of this clone revealed an ORF of 367 cod ons encoding a single domain type-F or family 10 enzyme, which was designat ed as XynA. Purification of the enzyme following over-expression in E. coli produced an enzyme of 42 kDa with a temperature optimum of 75 degrees C wh ich can efficiently bind and hydrolyse insoluble xylan. The pH optimum of t he enzyme is 6.5, but it is active over a broad pH range. A homology model of the xylanase has been constructed which reveals a series of surface arom atic residues which form hydrophobic clusters. This unusual structural feat ure is strikingly similar to the situation observed in the structure determ ined for the type-G xylanase from the Bacillus D3 strain and may constitute a common evolutionary mechanism imposed on different structural frameworks by which these xylanases may bind potential substrates and exhibit thermos tability. (C) 1999 Elsevier Science B.V. All rights reserved.