Part:BBa_K4382004

Glucuronoxylan depolymerase - Xylanase C

Usage

Xylanase C (Xyn C)[NC_000964.3] is an endoxylanase which is a part of the GH-5 family of glycosyl hydrolases. The main function of XynC is the production of methyl-glucuronic acid substituted xylan from larger xylan polymers.

Biology

The enzyme is most active at a temperature of around 65 degrees celsius and a pH of 6. Much like Xylanase A (Biobrick present already). this enzyme works on the substrate 4-O-methyl-D-glucuronoxylan (MeGAXn - also known as aldouronates).

Hydrolysis products of MeGAXn were predicted through MALDI TOF mass spectrometry. Two fractions were obtained - one was the filtrate fraction which was obtained upon centrifugation of the enzyme-substrate system. The other was the retentate - i.e. the residue remaining post centrifugation. Both fractions were analyzed using Mass spectrometry - which showed that the products formed included all Methyl Glucuronic acid substituted Xylans ranging from n = 4 to n = 20.

Further analysis of the products obtained was conducted by NMR - which showed that the MeGA substitution was present on the xylose which was penultimate to the XynC reducing end. Further analysis showed that the MeGAXn is cleaved at the xylosidic bond penultimate to that linking the xylose residue to that substituted with the MeGA.

In addition to this, the xylanases obtained from Bacillus subtilis 168 were fractionated and the products obtained were analyzed through thin layer chromatography. It was found that the fraction containing XynC gave various aldouronates, with xylobiose, xylotriose and other unsubstituted products being absent, while the unfractionated sample gave all these products in addition to MeGAX3. The XynA containing fraction has xylobiose, xylotriose and MeGAX4

In Bacillus subtilis 168 the enzyme is constitutively expressed - unlike other xylanases.

References

1. St. John, F. J., Rice, J. D., & Preston, J. F. (2006, December 15). Characterization of XynC from Bacillus subtilis subsp. subtilis Strain 168 and Analysis of Its Role in Depolymerization of Glucuronoxylan. Journal of Bacteriology, 188(24), 8617–8626. https://doi.org/10.1128/jb.01283-06

2. Biely, P., Malovíková, A., Uhliariková, I., Li, X. L., & Wong, D. W. (2015, July 26). Glucuronoyl esterases are active on the polymeric substrate methyl esterified glucuronoxylan. FEBS Letters, 589(18), 2334–2339. https://doi.org/10.1016/j.febslet.2015.07.019

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1120
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1120
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 762
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1120
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1120
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 295