Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, e ncoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino aci ds with a molecular weight of 115,564. Xyn10A is a multidomain enzyme compo sed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 car bohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. I mmunological analysis indicated the presence of Xyn10A in the culture super natant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn 10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that b oth the CBM and the SLH domains are fully functional in the recombinant enz yme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, reta ined the ability to bind to BMC. This observation suggests that the catalyt ic domain or the thermostabilizing domains are responsible for binding of t he enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was high ly active toward xylan but not toward p-nitrophenyl-beta -D-xylopyranoside pyranoside, p-nitrophenyl-beta -D-cellobioside, or carboxymethylcellulose.