Difference between revisions of "Part:BBa K4194019"
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The characteristic peaks determines that the product has dimerization products. | The characteristic peaks determines that the product has dimerization products. | ||
[[File:The characteristic peaks determines that the product has dimerization products..png|900px|thumb|left|]] | [[File:The characteristic peaks determines that the product has dimerization products..png|900px|thumb|left|]] | ||
| − | [[File:NMR of hydroxyhexadecanoic acid..png| | + | [[File:NMR of hydroxyhexadecanoic acid..png|600px|thumb|left|NMR of hydroxyhexadecanoic acid.]] |
| − | [[File:NMR of dimeric compounds.png| | + | [[File:NMR of dimeric compounds.png|600px|thumb|left|NMR of dimeric compounds.]] |
| + | [[File:NMR of trimeric compounds.png|600px|thumb|left|NMR of trimeric compounds.]] | ||
<br style="clear: both" /> | <br style="clear: both" /> | ||
| + | Integration comparison of the peaks of 2/16/20 C to determine the degree of aggregation. | ||
| + | [[File:Integration comparison of the peaks of 2,16,20 C to determine the degree of aggregation..png|600px|thumb|left|]] | ||
| + | (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, further confirming the better activity of the new FadD enzyme. | ||
| + | <br style="clear: both" /> | ||
| + | ==Gel Permeation Chromatography（GPC）== | ||
| + | [[File:Gel Permeation Chromatography（GPC）1.png|600px|thumb|left|]] | ||
| + | [[File:Gel Permeation Chromatography（GPC）2.png|600px|thumb|left|]] | ||
| + | <br style="clear: both" /> | ||
| + | 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. This further indicates that the efficiency and quality of the system to produce PHFA were improved after replacing FadD enzyme. 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. | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K4194019 parameters</partinfo> | <partinfo>BBa_K4194019 parameters</partinfo> | ||
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Revision as of 07:27, 30 September 2022
fadD
The gene codes for long chain acyl-CoA synthase(CoA ligase).
Usage and Biology
CoA ligase
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 195
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 504
Illegal XhoI site found at 1006 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 408
Illegal AgeI site found at 1033 - 1000COMPATIBLE WITH RFC[1000]
Experiment and result
Gas chromatographic analysis of hydrolyzed products
The BUCT-China team attempted to synthesize PHFA (polyhydroxyfatty acid, a biocompatible polymer material) using an enzyme system containing CoA Ligase.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.
NMR hydrogen spectrogram analysis of polymers
The characteristic peaks determines that the product has dimerization products.
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, further confirming the better activity of the new FadD enzyme.
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. This further indicates that the efficiency and quality of the system to produce PHFA were improved after replacing FadD enzyme. 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.