Part:BBa_K3599004

laNIT

Overview

Nitrilase coming from Labrenzia aggregata, catalysis of the reaction: nitrile + 2H₂O = carboxylate + NH₃. The codon usage was optimized for expression in Escherichia coli.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Primitive Species Background

A novel alphaproteobacterium containing bacteriochlorophylla, and a proposal for reclassification of Stappia aggregata as Labrenzia aggregata comb. nov., of Stappia marina as Labrenzia marina comb. nov. and of Stappia alba as Labrenzia alba comb. nov., and emended descriptions of the genera Pannonibacter, Stappia and Roseibium, and of the species Roseibium denhamense and Roseibium hamelinense.

Design & Experience

In iGEM2020 TPR_China Project, the laNIT was expressed using pTac system, which was pre-embedded into commercial expression vector pET-28b. The whole expression cassette can be found in BBa_K3599008.

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.

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.

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.

Standard curve of NH₄⁺

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.

Experimental phenomena of enzyme activity test

Enzymatic activity of nitrilase

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

Huihui, Sun, Wenyuan, Gao, Haiyang, & Fan, et al. (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. 10.1007/s10529-015-1830-4