Sequence, overproduction and purification of the family 11 endo-beta-1,4-xylanase encoded by the xyl1 gene of Streptomyces sp S38

The xyl1 gene encoding the Xyl1 xylanase of Streptomyces sp. strain S38 was cloned by screening an enriched DNA library with a specific DNA probe and sequenced. Three short 5 bp-CGAAA- sequences are located upstream of the St reptomyces sp. S38 xyl1 gene 105, 115 and 250 bp before the start codon. Th ese sequences, named boxes 1, 2 and 3, are conserved upstream of the Actino mycetales xylanase genes and are specifically recognized by a DMA-binding p rotein (Giannotta et al., 1994. FEMS Microbiol. Lett. 142, 91-97) and could be probably involved in the regulation of xylanase production. The Xyl1 OR F encodes a 228 residue polypeptide and the Xyl1 preprotein contains a 38 r esidue signal peptide whose cleavage yields a 190 residue mature protein of calculated M-r=20 585 and basic pI value of 9.12. The molecular mass of th e produced and purified mature protein determined by mass spectrometry (20 586 +/- 1 Da) and its pI (9.8) agree with these calculated values. Its N-te rminal amino-acid sequence confirmed the proposed cleavage site between the signal peptide and the mature protein. Comparisons between Xyl1 and the 62 other xylanases belonging to family 11 allowed the construction of a phylo genetic tree and revealed its close relationship with Actinomycetales enzym es. Moreover, nine residues were found to be strictly conserved among the 6 3 xylanases. (C) 1999 Elsevier Science B.V. All rights reserved.