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From 2014.igem.org

Introduction

Endo-1, 4-β-xylanases (in short Xylanases) are enzymes that hydrolyze the internal glycosidic bonds within the xylan backbone. Xylan is one major components of wood (accounting for approximately 20-35% of plant cell wall dry mass) and therefore one of the most abundant organic molecules in nature [1]. A variety of prokaryotes and eukaryotes, but not animals, express Xylanases to use the monosaccharides from Xylan. We used a recombinant XylA from Bacillus subtilis expressed in E. coli because it is a well-established model organism and the XylA because of its structural properties. Xylanases find broad application in industry, especially for bio-bleaching in pulp production, animal feedstocks and the production of bio-Ethanol. For these applications often more stable variants are needed and therefore the engineering of heat stable xylanases is an active research topic. Common approaches to this problem are to investigate potentially heat-stable xylanases fromthermophilic archae or bacteria and to introduce disulfide bonds in order to generate more robust xylanases.

We want to introduce intein-mediated circularization as an alternative approach to confer heat stabilityproteins. We chose the xylanase of Bacillus subtilis which is a promising enzyme to be used in pulp bleaching. Moreover, N- and C-terminus of this xylanase are in close (4.6 Å , see Figure X) which facilitates circularization. To achieve circularization we used split inteins . This method produces after the splicing reaction a scar of 6 amino acids, which can be bridge the gap between the native termini.

Experimental Procedures

Construct Names	Motivation
Circular	Most similar to native Xylanase, but by expected to be more stable because of circularization
Circular with His-Tag	Same as Circular plus the ability for easier purification and verifaction on Western Blot
Circular with His-Tag, Mutation in C-Ext. (Cys-> Gly)	Control to see a non-circularized form and have a sample to assess the degree the splicing reaction takes place.