Xylanase from the extremely thermophilic bacterium 'Caldocellum saccharolyticum': overexpression of the gene in Escherichia coli and characterization of the gene product

A xylanase encoded by the xynA gene of the extreme thermophile 'Caldocellum saccharolyticum' was overexpressed in Escherichia coli by cloning the gene downstream from the temperature-inducible λ p(R) and p(L) promoters of the expression vector pJLA602. Induction of up to 55 times was obtained by growing the cells at 42°C, and the xylanase made up to 20% of the whole-cell protein content. The enzyme was located in the cytoplasmic fraction in E. coli. The temperature and pH optima were determined to be 70°C and pH 5.5 to 6, respectively. The xylanase was stable for at least 72 h if incubated at 60°C, with half-lives of 8 to 9 h at 70°C and 2 to 3 min at 80°C. The enzyme had high activity on xylan and ortho-nitrophenyl β-D-xylopyranoside and some activity on carboxymethyl cellulose and para-nitrophenyl β-D-cellobioside. The gene was probably expressed from its own promoter in E. coli. Translation of the xylanase overproduced in E. coli seemed to initiate at a GTG codon and not at an ATG codon as previously determined.