Team:Lethbridge HS/Parts





Parts Overview






Our project this year involves the improvement of compost breakdown through the implementation of pectinase enzymes Pnl, PelB and PelC from P. amylolyticus. All of these parts are new to the registry. Additionally, each of these enzymes have also been fused to a GFP reporter. Each part developed contains a C-terminal His tag either on the synthesized gene or on the GFP reporter. Although we have also developed a thermostable variant it has not been placed onto the registry this year. For more information on this part please see our modelling Page.

Both the enzymes with and without the fusion partner can be found on the registry and our basic or composite pages. A more detailed explanation of these pectinases are seen below or feel free to click on the Registry BBa number to go to the parts page to learn more and learn about our results.


All of our composite parts were designed using the T7 promoter (BBa_I712074), medium strength RBS (BBa_J61100), and double terminator (BBa_B0014).





Pnl (BBa_K3349000)

Original organism: Paenibacillus amylolyticus.

Pnl is a ~35 kDa enzyme that cuts at methylated sites of the pectin [1]. This enzyme is unique as it has a high affinity for methylated sites. Pnl is used in combination with PelB and PelC to effectively cleave and therefore breakdown homogalacturonan substrates in terms of P. amylolyticus enzymes. Optimal reaction temperature is 55°C and optimal pH is between 9-10 [1].


PelB (BBa_K3349001)

Original organism: Paenibacillus amylolyticus.

PelB is a ~48 kDa Cuts at unmethylated sites of the pectin [1].This enzyme can be used in combination with Pnl and PelC to effectively cleave and therefore breakdown homogalacturonan substrates in terms of P. amylolyticus enzymes. This protein has been characterized to cleave methylated pectin (at unmethylated sites) and polygalacturonic acid [2]. The optimal temperature for PelB activity is 70°C [1]


PelC (BBa_K3349002)

Original organism: Paenibacillus amylolyticus.

PelC is ~43 kDa Cuts at the junction of methylated and unmethylated sites of the pectin [1].This enzyme can be used in combination with PelB and Pnl to effectively cleave and therefore breakdown homogalacturonan substrates in terms of P. amylolyticus enzymes.This protein has been characterized to cleave methylated pectin (at unmethylated sites) and polygalacturonic acid [2]. PelC has an optimal activity at 55°C and a pH of 10 [1].


References:

  1. Keggi, C and Doran-Peterson, J. (2020) The homogalacturonan deconstruction system of Paenibacillus amylolyticus 27C64 requires no extracellular Pectin Methylesterase and has significant industrial potential. Applied and environmental microbiology. 86, e02275-19.
  2. Boland, W., Henriksen, E., and Doran-Peterson, J. (2010) Characterization of two Paenibacillus amylolyticus strain 27C64 pectate lyases with activity on highly methylated pectin.