Cloning, expression, and sequence analysis of the gene encoding the alkali-stable, thermostable endoxylanase from alkalophilic, mesophilic Bacillus sp. Strain NG-27

Abstract

Alkalophilic Bacillus sp. strain NG-27 produces a 42-kDa endoxylanase active at 70 degrees C and at a pH of 8.4. The gene for this endoxylanase was cloned and sequenced. The gene contained one open reading frame of 1,215 bases. An active site characteristic of the family 10 beta-glycanases was recognized between amino acids 303 and 313, with the active glutamate at position 310. Though highly thermostable, the enzyme contains no cysteine residue.

FIG. 1

(A) Zymographic analysis of preparation of xylanase from Bacillus sp. strain NG-27 on xylan (lane 1) or CMX (lane 2). (B) Zymographic analysis of xylanase on Xylan. Lane 1, enzyme preparation from Bacillus sp. strain NG-27; lane 2, xylanase purified from E. coli DH5Δlac carrying the plasmid pGNG19. The positions of standard protein molecular weight markers (Sigma Chemical Co.) run on the same gel are indicated at left. The sizes are as follows: 66 kDa (bovine albumin), 45 kDa (egg ovalbumin), 36 kDa (glyceraldehyde-3-phosphate dehydrogenase), 29 kDa (carbonic anhydrase), and 24 kDa (trypsinogen).

FIG. 2

Mapping of the 5′ ends of the xylanase gene transcripts by primer extension. The partial nucleotide sequence of pGNG19 generated using Xyla1 primer is represented (lanes A, T, G, and C). The extension products of Xyla1 primer obtained by using total RNA from E. coli carrying pGNG19 plasmid and Bacillus sp. strain NG-27 are shown (lanes E and B, respectively). The nucleotide sequence surrounding the transcription initiation sites is shown, and the −10 sequence motif is boxed. The numbers −131 and −132, in parentheses and highlighted by asterisks, indicate the transcription initiation sites for the xylanase gene when expressed in Bacillus and E. coli.

FIG. 3

CLUSTAL V-based alignment of the sequence of the thermostable, alkalistable xylanase from the mesophilic Bacillus species strain NG-27 (BAC.SP.NG-27) with sequences of xylanases from related Bacillus species (XYNA_BACS5), thermophilic microbes (XYN1_BACST, CEXY_CLOSR, XYNB_THENE), and mesophilic microbes (XYNA_ASPAK, CELLVIB). Single, fully conserved residues are boxed, and strong or weak conservation of residue type is indicated by dark or light shading, respectively.