We decided to use nitrilase - a protein that degrades nitriles - for degradation of PAN.
According to the literature, we selected nitrilase with high enzymatic activity and relatively complete sequences which are NhNIT（BBa_K3599002）, AdNIT（BBa_K3599003）,PfNIT(BBa_K3599005) ，AfNIT(BBa_K3599006)and LaNIT (BBa_K3599004).
1. Selection criteria of NIT
The selected criteria of our team for nitrile hydrolase include following steps: First, the coding sequence of nitrilase should be reported; Secondly, it needs to be done in the E.coli heterogenic expression, and there are clear conditions for expression; Third, its activity has been tested, the enzyme activity is high (above 40U/mg), and there are clear test conditions; Finally, the degradation ability for PAN should be tested.
Based on those standards, we read a lot of literature and finally selected five genes: NhNIT, AdNIT, LaNIT, PfNIT and AfNIT.
2. Information about the selected NIT:
|Name||Organism||Absolute Activity||Function in the Regional Species|
|NhNIT||NhNITNectria haematococcaAdNITAchromobacter denitrificans||111.1 U/mg||arylacetonitrilase activities |
|AdNIT||Achromobacter denitrificans||70.01 U/mg||catalyze the hydrolysis of organic cyanides or nitriles (structural formula is R-CN) with the formation of the corresponding acids and ammonium |
|LaNIT||Labrenzia aggregata||89.2U/mg||nitrogen compound metabolic process |
|PfNIT||Pseudomonas fluorescens||67.65 U/mg||to use different arylacetonitriles (e.g. 2-phenylpropionitrile) as nitrogen sources and converted the nitriles to the corresponding α-substituted carboxylic acids |
|AfNIT||Alcaligenes faecalis||41.69 U/mg||to convert chemically reactive and harmful nitriles directly into carboxylic acids as the sole product under mild conditions |
3. Enzymatic Activity
We successfully constructed 3 NITs which are NhNIT, AdNIT,our effector system is construct on pET28-b plasmid and bind with upstream LacI sequence, and LaNIT and tested the enzymatic activity of La & AdNITs.
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.
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 from Fig. 4, 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,and it verified that AdNIT (BBa_K3599003) and LaNIT (BBa_K3599004) could express enzyme activity better.