Part:BBa_K4641000

TccA

Ochrobactrum sp. TCC-2 TccA amidase protein coding region. This part codes for an amidase involved in cleaving the amide bonds in N-(4-Chlorophenyl)-N-(3,4-dichlorophenyl)urea (Common name: Triclocarban or 3,4,4-Trichlorocarbanilide). The breakdown forms two daughter compounds : 3,4-Dichloroaniline and 4-Chloroaniline

Profile

Name: TccA
Base Pairs: 1452 bp
Origin: Ochrobactrum TCC-2
Properties: Amidase involved in cleaving the amide bonds in 3,4,4-Trichlorocarbanilide

Usage and Biology

Aniline compounds are among the hardest compounds to biodegrade due to the high stability of the carbon-nitrogen bond. However, they are a component in many micropollutants in wastewater. Triclocarban(3,4,4′-Trichlorocarbanilide)(TCC) is one such pollutant. One of the major objectives of our project was to biodegrade TCC to non toxic byproducts. TccA gene, from the Ochrobactrum sp. TCC-2, codes for a novel amidase enzyme(EC 3.5.1.4), responsible for the hydrolysis of the two amide bonds of TCC and its dehalogenated analogs 4,4'-dichlorocarbanilide (DCC) and carbanilide (NCC) to more biodegradable chloroaniline or aniline products. TccA also has hydrolytic activity to a broad spectrum of amide bonds from herbicides, insecticides and chemical intermediates. The broad substrate spectrum of TccA suggested potential use for bioremediation applications. [1]
In our project, TccA amidase is used as an agent to biodegrade the TCC present in the sludge of the wastewater treatment plant.

Cultivation and Purification

Shake Flask Cultivations

The first trials to produce TccA amidase were carried out in a 250mL conical flask at 37 degrees Celsius.

• flask design: shake flask at 180rpm
• medium: IPTG induction in LB broth
• antibiotics: 100 µg mL-1 Ampicillin
• temperature: 37 °C
• cultivation time: 48 h

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 237
  Illegal XhoI site found at 1423
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]