Cloning, expression and sequence analysis of the gene encoding an endoxylanase from Bacillus halodurans MIR32

MARTINEZ M. ALEJANDRA; DELGADO OSVALDO D.; SINERIZ FAUSTINO; HATTI-KAUL RAJNI

Naples, Italy

Congreso; 1st INTERNATIONAL CONGRESS ON EXTREMOPHILES; 2002

The International Society for Extremophiles (ISE)

Bacillus halodurans MIR32 was isolated from soil samples in Argentina as an excellent xylan degrader [1,2]. The gene (XynNt) coding for an extracellular endoxylanase [3] from the microorganism was amplified using PCR techniques. Full sequence of the gene was analysed and putative promoter elements, Shine-Dalgarno site and leader peptide sequences were identified. Properties of XynNt based on the primary structure analysis were predicted using the ProSite and SignalP tools on the ExPASy server (http://www.expasy.ch/tools). Based on the amino acid identity and biochemical characteristics, we determined that XynNt is an endo-1,4--xylanases belonging to family 11 of glyscosyl hydrolases. The gene xynNt encodes a protein of 210 amino acids with calculated molecular mass of 23 kDa and an isoelectric point of 10. These data were coincident with experimental data previously obtained [3].Sequence corresponding to the mature peptide was subcloned into the pET22b+ (Novagene) expression vector resulting in pNX33 construction, which contains a His6 tag at the C-terminal terminus. High-level expression of xylanase by BL21 DE3 pLYS E. coli host harbouring the construction was observed upon induction with IPTG.Purification of the recombinant B. halodurans endoxylanase was facilitated by immobilized metal ion affinity chromatography, resulting in 80-fold purification and 72 % yield.