Influence of the Transposition of the Thermostabilizing Domain of Clostridium thermocellum Xylanase (XynX) on Xylan Binding and Thermostabilization

A xylanase gene, xynX, of Clostridium thermocellum had one thermostabilizing domain (TSD) between the signal peptide sequence and the catalytic domain (CD). The TSD of a truncated xylanase gene, xynX′(TSD-CD), was transpositioned from the N terminus to the C terminus of the CD by overlapping PCRs, and a modified product, xynX′(CD-TSD), was constructed. XynX′(TSD-CD) had a higher optimum temperature (70°C versus 65°C) and was more thermostable (residual activity of 68% versus 46% after a 20-min preincubation at 70°C) than the one without the TSD, XynX′(CD). However, the domain-transpositioned enzyme, XynX′(CD-TSD), showed a lower optimum temperature (30°C) and thermostability (20%) than XynX′(CD). Both XynX′(TSD-CD) and XynX′(CD-TSD) showed significantly higher binding capacity toward xylan than XynX′(CD), and the domain transposition did not cause any change in the binding ability. XynX′(TSD-CD) and XynX′(CD-TSD) also showed considerable binding to lichenan but not to carboxymethyl cellulose and laminarin. XynX′(TSD-CD) and XynX′(CD-TSD) had higher activities for insoluble xylan than XynX′(CD), while XynX′(CD) was more active against soluble xylan than XynX′(TSD-CD) and XynX′(CD-TSD). These results indicate that the TSD of XynX has dual functions, xylan binding and thermostabilization, and the domain should also be classified as a xylan-binding domain (XBD). The binding capacity of the XBD was not affected by domain transpositioning within the gene.