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Influence of transposition and insertion of additional binding domain on expression and characteristics of xylanase C of Clostridium thermocellum
- Khan, M. Imran Mahmood, Sajjad, Muhammad, Ali, Imran, Ahmad, Sajjad, Akhtar, M. Waheed
- Journal of biotechnology 2010 v.150 no.1 pp. 1-5
- Clostridium thermocellum, Escherichia coli, active sites, biotechnology, carbohydrate binding, cellulosome, genes, oats, plasmids, protein binding, proteins, thermal stability, xylan, xylanases
- Clostridium thermocellum encodes a xylanase gene (xynC) which is the major component of its cellulosome. XynC is a multidomain enzyme comprising of a substrate binding domain at the N-terminal followed by the catalytic domain and a dockerin domain. To study the influence of binding domain on activity, stability and expression of the enzyme the protein with the binding domain at C-terminal (XynC-CB), and the one with the binding domain at both N- and C-terminal (XynC-BCB) were expressed in E. coli. Recombinant plasmids, pXynC-CB and pXynC-BCB were constructed by inserting the corresponding gene in pET22b(+). XynC-CB and XynC-BCB were expressed at levels around 30% and 33% of the total E. coli cell proteins, respectively, while losing 40% and 20% of their activities at 70°C for 120min, respectively. The specific activities of XynC-CB, XynC-BCB were 76 and 98Umg⁻¹, while the activities on equimolar basis were 4410 and 7450UμM⁻¹ against birchwood xylan, respectively. Their overall activities produced in the culture were 3660 and 5430UL⁻¹ OD₆₀₀ ⁻¹. Substrate binding studies showed that in case of XynC-C 51% of the activity remained unbound to birchwood xylan, whereas in the cases of XynC-BC, XynC-CB and XynC-BCB the activities left unbound were 33%, 32% and 12%, respectively, under the assay conditions used. Similar binding values were obtained in the case of oat spelt xylan. K m values for XynC-CB and XynC-BCB against birchwood xylan were found to be 3.1 and 1.47mgml⁻¹, respectively. Thus addition of a second carbohydrate binding domain at the C-terminal of the catalytic domain enhances activity, substrate affinity as well as thermostability.