Part:BBa_K3599003
adNIT
Overview
Nitrilase coming from Achromobacter denitrificans, catalysis of the reaction: nitrile + 2H2O = carboxylate + NH3. The codon usage was optimized for expression in Escherichia coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 629
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 191
Illegal BsaI site found at 374
Primitive Species Background
The type species of gram negative, aerobic bacteria in the genus. A genus of strictly aerobic, non-spore forming rods. Soil and water are regarded as the natural habitat. They are sometimes isolated from a hospital environment and humans. Previously in the genus Alcaligenes, the classification and nomenclature of this species has been frequently emended. The two subspecies, Achromobacter xylosoxidans subsp. denitrificans and Achromobacter xylosoxidans subsp. xylosoxidans are associated with infections.
Design & Experience
In iGEM2020 TPR_China Project, the adNIT was expressed using pTac system, which was pre-embedded into commercial expression vector pET-28b. The whole expression cassette can be found in BBa_K3599009.
SDS-PAGE Verification of Nitrilase
We performed SDS-PAGE verification on the fermentation broth of the engineered strain after induction, and the results are shown below.
Electrophoresis results show that our sample has a specific band around 31KD compared to the negative control, this proves that the engineered bacteria we constructed have expressed nitrilase protein.
Nitrilase Enzyme Activity
The nitrilase has PAN as substrate to degrade PAN into phenylacetic acid and ammonium. Ammonium and Nessler's Reagent can produce reddish-brown precipitation. The shade is proportional to the concentration of NH4+ produced by PAN degradation.
I. Standard Curve
We plotted the standard curve of the ammonium concentration. The experimental sample's NH4+ concentration can thus be calculated from the standard concentration equation.
II. Nitrilase Enzyme Activity
The activity of nitrilase was verified by detecting the ammonium ions produced by the degradation of PAN by the bacterial liquid. We define U as the amount of PAN that can be degraded per microliter per hour.
As can be seen, the U value of pTac-laNIT is 0.6115, which is 1.27 times that of the negative control;the U value of pTac-adNIT is 0.6205, which is 1.3 times that of the negative control.These data indicate that our engineered bacteria have high activity of nitrilase.
Reference
Novikov, A. D. , Riabchenko, L. E. , Leonova, T. E. , Larikova, G. A. , & Yanenko, A. S. . (2016). A bacterial strain alcaligenes denitrificans С-32 contains two nitrilases with different substrate specificities. 10.21519/0234-2758-2016-32-6-45-52
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