Part:BBa_K5117003

AtCelA

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

AtCelA 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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Enzyme characterization according to literature

In the study by Weng et al. (2022), titled "Immobilization of recombinant endoglucanase (CelA) from Clostridium thermocellum on modified regenerated cellulose membrane", the researchers focused on enhancing the industrial applicability of cellulases through enzyme immobilization (Weng et al. 2022).

The gene encoding endoglucanase CelA from the cellulosome of Clostridium thermocellum was cloned into the plasmid pET21b-CelA-his. This construct was transformed into E. coli strains ER2566 and BL21. Successful cloning was confirmed via PCR, which displayed a target gene fragment of approximately 1.6 kb. Protein expression analysis revealed that E. coli ER2566, induced at 37 °C for 6 hours, produced the highest amount of CelA. SDS-PAGE analysis showed a prominent band at around 60 kDa, confirming the expression of the target protein (Weng et al. 2022).

The goal of this work was to improve the stability and reusability of CelA. Therefore, the enzyme was immobilized on modified regenerated cellulose (RC) membranes. RC membranes were modified to incorporate cobalt ions (RC-EPI-IDA-Co²⁺) for coordination coupling. RC membranes were modified to develop aldehyde functional groups (RC-EPI-DA-GA) for covalent bonding with the enzyme (Weng et al. 2022).

The free enzyme exhibited maximum activity at pH 5, while the immobilized enzymes showed optimal activity at pH 6. Immobilized CelA maintained 80 – 60 % relative activity across a wide pH range (pH 4 – 9), indicating greater pH stability compared to the free enzyme. The optimal temperature for the free enzyme and RC-EPI-IDA-Co²⁺-CelA was 60 °C, while RC-EPI-DA-GA-CelA displayed an optimal temperature of 70 °C. Immobilized enzymes demonstrated superior thermostability, retaining 85 % relative activity within the temperature range of 50 – 70 °C. At higher temperatures (80 – 90 °C), immobilized enzymes retained significantly more activity than the free enzyme, highlighting improved thermal resistance due to immobilization (Weng et al. 2022).

After 15 days of storage at 4 °C, immobilized CelA retained 80 % of its relative activity, demonstrating good storage stability. After five reuse cycles, RC-EPI-IDA-Co²⁺-CelA and RC-EPI-DA-GA-CelA retained 63 % and 53 % of their initial activity, respectively. The immobilization of recombinant CelA on modified regenerated cellulose membranes enhanced the enzyme's thermal stability, pH tolerance, and reusability (Weng et al. 2022).

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

• Beguin P., Cornet P., Aubert J. P. (1985): Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum. Journal of bacteriology 162(1), 102-105. https://doi.org/10.1128/jb.162.1.102-105.1985
• Van Der Veen D., Lo J., Brown S. D., Johnson C. M., Tschaplinski T. J., Martin M., ___ Lynd L. R. (2013): Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways. Journal of Industrial Microbiology and Biotechnology 40(7), 725-734. https://doi.org/10.1007/s10295-013-1275-5
• Schmidt, A. et al., Endoglucanase A from Clostridium thermocellum at atomic resolution (2001) https://doi.org/10.2210/pdb1IS9/pdb
• Schmidt A., Gonzalez A., Morris R. J., Costabel M., Alzari P. M., Lamzin, V. S. (2002): Advantages of high-resolution phasing: MAD to atomic resolution. Acta Crystallographica Section D: Biological Crystallography 58(9), 1433-1441. https://doi.org/10.1107/S0907444902011368
• Kitago Y. et al., Crystal structure of Cel44A, GH family 44 endoglucanase from Clostridium thermocellum (2006) https://doi.org/10.2210/pdb2E4T/pdb
• Kitago Y. et al., Crystal structure of Cel44A, GH family 44 endoglucanase from Clostridium thermocellum (2007) https://doi.org/10.2210/pdb2EEX/pdb
• Kitago Y. et al., The crystal structure of Cel44A (2011) https://doi.org/10.2210/pdb2E0P/pdb
• Kitago Y., Karita S., Watanabe N., Kamiya M., Aizawa T., Sakka K., Tanaka I. (2007): Crystal structure of Cel44A, a glycoside hydrolase family 44 endoglucanase from Clostridium thermocellum. Journal of Biological Chemistry 282(49), 35703-35711. https://doi.org/10.1074/jbc.M706835200
• Gene sequence: https://www.ncbi.nlm.nih.gov/nuccore/K03088
• Protein sequence: https://www.ncbi.nlm.nih.gov/protein/AAA83521
• UniProtKB: https://www.uniprot.org/uniprotkb/A3DC29/entry

References

Weng Z. H., Nargotra P., Kuo C. H., Liu Y. C. (2022): Immobilization of recombinant endoglucanase (CelA) from Clostridium thermocellum on modified regenerated cellulose membrane. Catalysts 12(11), 1356. https://doi.org/10.3390/catal12111356