Coding

Part:BBa_K1175005

Designed by: Nicholas K. Goldner   Group: iGEM13_WLC-Milwaukee   (2013-09-27)

endo-1,4-beta-xylanase xynA from Bacillus Subtilis Subtilis 168

The endo-1,4-beta-xylanase gene xynA cleaves xylan polysaccharide chains to form shorter xylan chains. This gene has been isolated from the bacterium Bacillus subtilis subtilis 168.

Usage and Biology

Xylan is a molecule similar to cellulose, and after cellulose the most abundant biomass material on earth. It is a major structural component of plant cell walls. Furthermore, xylan crosslinks with cellulose and other cell wall components, inhibiting access of cellulases (1). Xylose is the sugar monomer of xylan as glucose is to cellulose. Xylose cannot be used in the human body as a source of energy. Endo-1,4-beta-xylanase (xynA) breaks the xylan chains into shorter chains, and may be stearically hindered by side chains (2).

A beta-xylanase such as Endo-1,4-beta xylanase may be used to degrade xylan to facilitate cellulase activity. Another use may be in conjunction with an exo-xylanase to efficiently break down xylan into xylose monomers (a pentose sugar).

Enzymatic Activity of Gene Product

As stated above, the function of the gene product is xylan degredation. The enzyme's catabolic activity results from endohydrolysis of 1,4-beta-D-xylosidic linkages in xylan molecules (4).

(1) http://newscenter.lbl.gov/feature-stories/2012/11/12/a-better-route-to-xylan/ (2) http://www.nutrex.be/sites/default/files/wysiwyg-upload/nutrase-xyla-nsp-enzyme.pdf (3) http://subtiwiki.uni-goettingen.de/wiki/index.php/XynA (4) http://www.uniprot.org/uniprot/P18429

xynA

The endo-1,4-beta-xylanase xynA is a globular protein that has two residues of interest the nucleophile and acid-base cleavage sites at the E residues 78 and 172 highlighted in red.

xynA Enzyme Activity

This graph depicts the inhibition of the gene product of XynA found in Bacillus Subtillis Subtillis 168 (BsX) in comparison to the inhibition of the XynA found in Aspergillus Niger (AsX). Sorensen and Sibbensen were observing the inhibitory effects of the TAXI (Triticum Aestivum Xylanase Inhibitor) , specific to Glycoside hydrolase family 11 (GH 11), and XIP (xylanase inhibitor protein), specific to fungal GH 11 but not bacterial GH 11. which XynA is a member. Inhibition was tested with either pure XIP (BsX-XIP and AnX-XIP) or both XIP and TAXI ( BsX-Inhibitor Prep and AnX-Inhibitor Prep. It is evident from this graph that BsX is not effected by XIP but is strongly inhibited by TAXI, causing a decrease in residual xylanase activity by approximately 80%.

This graph depicts the effect of pH on the interaction between the BsX xylanase and the inhibitor TAXI at a 1:5 concentration. The pH profile of the of the inhibition resembles the ph profile of the enzyme, indicating that TAXI is a competitive inhibitor for the BsX Xylanase. This graph also depicts the optimal pH for the XynA enzyme from Bacillus Subtillis Subtillis 168 to be around 5.5.

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Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 85
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 489
    Illegal SapI.rc site found at 543


Improvement

The iGEM Team Heidelberg 2014 improved this part by removing the first 84 basepairs and adding a Ribosome Binding Site thus making the gene product (xylanase) retainable in the cytoplasm in E. coli.

The first 84 bases of the gene code for a signal peptide 1, 2 that is important for the secretion of the gene product into the extracellular medium in B. subtilis. This signal peptide was shown to be recognized and functional in E. coli as well 1 . After secretion, the signal peptide is removed and the mature protein, therefore, consists of the remaining 185 amino acids. In some applications, it is desirable to prevent secretion of the protein into the extracellular medium (for instance, for our own purposes of comparing the activity of linear and circular xylanase, we had to remove the signal peptide since it would not be exposed in the circular protein and the two proteins needed to be identical in their amino acid composition and localization). By eliminating the signal peptide and adding a Ribosome Binding Site (RBS) in our new part BBa_K1362020 we provide a new option for future users of xylanase.

Characterization

Activity after Heatshock assay for 30 minutes of linear Xylanase
Activity after Heatshock assay for 30 minutes of circular Xylanase
Fig. 3: Activity after Heatshock assay for 5 minutes of linear and circular Xylanase


We determined the activity of the linear and the circular Xylanase after Heatshock at different temperatures. Firstly the Xylanases were incubated at 37 °C, 50 °C, 56 °C, 60 °C and 66 °C. After 30 minutes substrate (EnzChek® Ultra Xylanase Assay Kit) was added and the fluorescence was measured for 150 minutes in a platereader at 37 °C. The results indicate a reduction of activity at temperatures higher than 50 °C and complete loss of function for temperatures higher than 60 °C.

For further characterization and as another control the 2 constructs were again expressed and tested in a shorter heatshock assay (5 minutes at 63 °C). The results in Fig. 3 show an increased heatstability of the circular xylanase compared to the linear after heatshock, which shows no more activtiy.

References

1. Is Helianti, Niknik Nurhayati, Maria Ulfah, Budiasih Wahyuntari, and Siswa Setyahadi, “Constitutive High Level Expression of an Endoxylanase Gene from the Newly Isolated Bacillus subtilis AQ1 in Escherichia coli,” Journal of Biomedicine and Biotechnology, vol. 2010, Article ID 980567, 12 pages, 2010. doi:10.1155/2010/980567

2. http://www.uniprot.org/uniprot/P18429

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