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Influence of transposition and insertion of additional binding domain on expression and characteristics of xylanase C of Clostridium thermocellum

Paper ID Volume ID Publish Year Pages File Format Full-Text
24182 43504 2010 5 PDF Available
Title
Influence of transposition and insertion of additional binding domain on expression and characteristics of xylanase C of Clostridium thermocellum
Abstract

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 120 min, respectively. The specific activities of XynC-CB, XynC-BCB were 76 and 98 U mg−1, while the activities on equimolar basis were 4410 and 7450 U μM−1 against birchwood xylan, respectively. Their overall activities produced in the culture were 3660 and 5430 U L−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. Km values for XynC-CB and XynC-BCB against birchwood xylan were found to be 3.1 and 1.47 mg ml−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.

Keywords
Clostridium thermocellum; Xylanase; Carbohydrate binding domain; Expression; Activity; Thermostability
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Influence of transposition and insertion of additional binding domain on expression and characteristics of xylanase C of Clostridium thermocellum
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 150, Issue 1, 1 October 2010, Pages 1–5
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us