Project Details
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.
|