Part:BBa_K5117004

AtCelG

This part contains the celG gene of Acetivibrio thermocellus (synonym Clostridium thermocellum) including its native signal peptide for secretion, encoding an endoglucanase (EC 3.2.1.4).

AtCelG only served for design purposes of the TU Dresden iGEM 2024 Team and was required for the construction of composite parts (see Contribution page).

Target organism: Bacillus subtilis

Main purpose of use: Gene expression and protein production using the host Bacillus subtilis

Design

For compatibility with the BioBrick RFC[10] standard, the restriction sites EcoRI, XbaI, SpeI, PstI and NotI were removed from the coding sequence. To make the part compatible with the Type IIS standard, BsaI and SapI sites were removed as well. This was achieved by codon exchange using the codon usage table of Bacillus subtilis (Codon Usage Database Kazusa).

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 1539
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 76
  
• 1000
  COMPATIBLE WITH RFC[1000]

Enzyme characterization according to literature

In the study by Lemaire and Béguin (1993), titled "Nucleotide sequence of the celG gene of Clostridium thermocellum and characterization of its product, endoglucanase CelG", the researchers determined the nucleotide sequence of the celG gene from Clostridium thermocellum. This gene encodes the enzyme endoglucanase CelG, which is involved in the degradation of cellulose (Lemaire & Béguin 1993).

The celG gene spans 1,698 base pairs, encoding a polypeptide of 566 amino acids with a molecular weight of approximately 63.1 kDa. The enzyme acts as a typical endoglucanase, causing a significant decrease in the viscosity of Carboxymethylcellulose (CMC) solutions while releasing a relatively low amount of reducing sugars. This suggests that CelG randomly cleaves internal β-1,4-glycosidic bonds in cellulose chains. CelG exhibits activity toward substrates such as carboxy-methyl p-nitrophenyl cellulose (CNPS) and p-nitrophenyl β-D-cellobioside (PNPC). The Michaelis constant for CNPC was found to be twice that for PNPC, while the maximum velocity was 40 times higher for CNPC, indicating a higher catalytic efficiency with CNPC (data not shown in publication) (Lemaire & Béguin 1993).

The enzyme exhibits optimal activity at a pH of 5.5 when assayed with CNPC, aligning with the acidic environments where cellulose degradation often occurs. CelG demonstrates considerable thermal stability. Less than 10% inactivation after 2 hours of incubation at 60 °C (data not shown in publication). Its ability to degrade cellulose efficiently at elevated temperatures and acidic pH makes it a valuable enzyme for industrial applications (Lemaire & Béguin 1993).

More information related to this part can be found in the following publications and databases:

• Gene sequence: https://www.ncbi.nlm.nih.gov/nuccore/X69390
• Protein sequence: https://www.ncbi.nlm.nih.gov/protein/CAA49187
• UniProtKB: https://www.uniprot.org/uniprotkb/Q05332/entry

References

Lemaire M. & Béguin P. (1993): Nucleotide sequence of the celG gene of Clostridium thermocellum and characterization of its product, endoglucanase CelG. Journal of bacteriology 175(11), 3353-3360. https://doi.org/10.1128/jb.175.11.3353-3360.1993