Part:BBa_K4194020

WS2

WS2 encodes waxy synthetase, which has a wide range of substrates and has a good affinity with medium-long chain ester acyl CoA and medium-long chain alcohols.

Usage and Biology

WS2 As an acyltransferase, the acyl group in the hydroxy fatty acid CoA replaces the H in hydroxyl group of the hydroxy fatty acid, and CoA-SH is generated at the same time.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 79
  Illegal PstI site found at 604
  Illegal PstI site found at 1003
  Illegal PstI site found at 1045
  Illegal PstI site found at 1108
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 79
  Illegal PstI site found at 604
  Illegal PstI site found at 1003
  Illegal PstI site found at 1045
  Illegal PstI site found at 1108
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 83
  Illegal BglII site found at 947
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 79
  Illegal PstI site found at 604
  Illegal PstI site found at 1003
  Illegal PstI site found at 1045
  Illegal PstI site found at 1108
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 79
  Illegal PstI site found at 604
  Illegal PstI site found at 1003
  Illegal PstI site found at 1045
  Illegal PstI site found at 1108
  Illegal AgeI site found at 697
  Illegal AgeI site found at 1306
  Illegal AgeI site found at 1354
  
• 1000
  COMPATIBLE WITH RFC[1000]

Experiment and result

The BUCT-China team attempted to synthesize PHFA (polyhydroxyfatty acid, a biocompatible polymer material) using an enzyme system containing waxy synthetase.

Gas chromatographic analysis of hydrolyzed products

The obtained product was hydrolyzed and then analyzed by gas chromatography, and the result analysis showed that the monomer of the polymer was hydroxy hexadecanoic acid. Therefore, it can be seen that the system carried out hydroxylation reaction and subsequent polymerization reaction on the fatty acid of the substrate 16C as expected, and the system basically met our requirements.

Results of gas chromatographic analysis of product hydrolysis.

NMR hydrogen spectrogram analysis of polymers

The above fig. shows the NMR of the simulated dimer product and the below fig. shows the NMR of the product.

The characteristic peaks determines that the product has dimerization products.

NMR of hydroxyhexadecanoic acid.

NMR of dimeric compounds.

NMR of trimeric compounds.

Integration comparison of the peaks of 2/16/20 C to determine the degree of aggregation.

(No. 16 C: No. 2 C) = 1.12: 0.13 = 8.61 (polymerization degree of 7-8)

(No. 20 C: No. 2 C) = 0.94: 0.19 = 7.23 (polymerization degree of 7-8)

(No. 16 C: No. 20 C) = 1.12: 0.94 = 1.19 (approximately equal to 6/5 = 1.2, polymerization degree of 5-6)
It could be judged that the degree of polymerization degree of the products should be between 5 and 8. Therefore, we determine that the system we designed is able to produce polyhydroxy fatty acids with a certain degree of polymerization.

Gel Permeation Chromatography（GPC）

We performed gel chromatography on the products, and the results showed that the number average molecular weight (Mn) was the highest at 198588, and the peak molecular weight (Mp) was 191,439. The molecules with molecular weights around this value accounted for the largest proportion of the overall material, which then indicates that the system has a higher proportion of large molecules with a higher degree of polymerization.Meanwhile, the polydispersity coefficient of this gel chromatography is about 1.258 which is closed to 1, which indicates that the substances are more uniformly dispersed during this assay and the results are highly reliable.