Team:TPR China/Collection

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Nitrilase Family

Overview

Name Number
AdNIT BBa_K3599003
LaNIT BBa_K3599004
NhNIT BBa_K3599002
PfNIT BBa_K3599005
AfNIT BBa_K3599005
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 [1]
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 [2]
LaNIT Labrenzia aggregata 89.2U/mg nitrogen compound metabolic process [4]
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 [5]
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 [6]

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.
Fig.1 Biosensor circuit of nitrilase
We performed SDS-PAGE verification on the fermentation broth of the engineered strain after induction, and the results are shown below.
Fig.2 The SDS-PAGE result of fermentation broth
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.
Fig.3 Experimental phenomena of enzyme activity test
Fig.4 Enzymatic activity of Nitrilase
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.

Reference:

[1]Veselá, A. B., Petříčková, A., Weyrauch, P., & Martínková, L. (2013). Heterologous expression, purification and characterization of arylacetonitrilases fromNectria haematococcaandArthroderma benhamiae. Biocatalysis and Biotransformation, 31(1), 49–56. https://doi.org/10.3109/10242422.2012.758117 [2]Novikov, A. D., Riabchenko, L. E., Leonova, T. E., Larikova, G. A., Lavrov, K. V., Glinskii, S. A., & Yanenko, A. S. (2016). A Bacterial Strain Alcaligenes denitrificans С-32 Contains Two Nitrilases with Different Substrate Specificities. Biotekhnologiya, 32(6), 45–52. https://doi.org/10.21519/0234-2758-2016-32-6-45-52 [3]Sun, H., Gao, W., Fan, H., Wang, H., & Wei, D. (2015). Cloning, purification and evaluation of the enzymatic properties of a novel arylacetonitrilase from Luminiphilus syltensis NOR5-1B: a potential biocatalyst for the synthesis of mandelic acid and its derivatives. Biotechnology Letters, 37(8), 1655–1661. https://doi.org/10.1007/s10529-015-1830-4 [4]UniProt ConsortiumEuropean Bioinformatics InstituteProtein Information ResourceSIB Swiss Institute of Bioinformatics. (2020, October 7). Nitrilase. https://www.uniprot.org/uniprot/A0P008. [5]Kiziak, C., Conradt, D., Stolz, A., Mattes, R., & Klein, J. (2005). Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme. Microbiology, 151(11), 3639–3648. https://doi.org/10.1099/mic.0.28246-0 [6]Liu, Z.-Q., Dong, L.-Z., Cheng, F., Xue, Y.-P., Wang, Y.-S., Ding, J.-N., … Shen, Y.-C. (2011). Gene Cloning, Expression, and Characterization of a Nitrilase fromAlcaligenes faecalisZJUTB10. Journal of Agricultural and Food Chemistry, 59(21), 11560–11570. https://doi.org/10.1021/jf202746a