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Xylanases (1,4-b-D-xylanhydrolase, EC 3.2.1.8) are endo-enzymes which randomly hydrolyse the b-1,4-xylose glycosidic  linkages of xylans, the principal polysaccharide of hemicelluloses, to release xylo-oligosaccharides. Xylanases are the focus of research efforts to harness this catalytic function since they have a potential application as an environmentally acceptable alternative to chlorine treatment of wood pulp in the Kraft process.

The gene for the class G/11xylanase A (XynA) from Bacillus subtilis strain 1A1 encodes a 185 amino acid protein, and has been amplified from total B. subtilis 1A1 genomic DNA by PCR using oligonucleotides that are complementary to the 5' and 3' non-coding regions of the xynA locus in the B. subtilis genomic sequence. The amplified fragment was cloned into the vector pT7T3 to give the vector pT7XyA, and DNA sequencing confirmed the expected construct.

After transfomation of E. coli DH5a with the pT7XyA, heterologous protein was constitutively produced and could be purified by cation-exchange chromatography from culture supernatants of E. coli cultures. Mass spectroscopy together with N-terminal sequencing by automated Edman degradation confirmed the correct post-translational processing of the secreted recombinant protein.

The coding region of the XynA gene was subjected to random mutagenesis using error prone PCR (epPCR), and the resulting random library was screened by selection from 9000 bacterial colonies using Congo Red staining on 1% xylan agar plates after incubation at 65oC. Of 90 colonies selected, 16 expressed thermostable XynA mutants, and amino acid sequence analysis that all these mutants were combinations of 6 discrete amino acid substitutions. Mapping of these positions in the crystal structure of xynA revealed that all mutants are located in a single domain of the protein (finger domain), suggesting that although multiple physico-chemical factors are involved in determining thermostability, the structural context through which these forces act is also highly relevant.