Part:BBa_K5427004
CelCD
Background Information
CelCD is a cellulase enzyme notable for its efficient secretion mechanism, driven by a conserved N-terminal sequence. This feature facilitates its extracellular excretion by E. coli, making CelCD highly valuable for industrial applications that favour external enzymatic activity. The enzyme's ability to hydrolyze carboxymethylcellulose (CMC) has been thoroughly investigated, with activity confirmed using cost-effective methods such as Congo red staining and spectrophotometric analysis. These characteristics make CelCD particularly appealing for industries focused on cellulose degradation, including biotextile processing and biofuel production, where effective and economical enzyme tracking is essential. Our selection of CelCD for this project is based on its established and standardized cloning strategy, similar to the rationale behind the earlier choice of Keratin31. This mature cloning protocol, combined with the enzyme's inherent secretion properties, simplifies both expression and purification, streamlining experimental workflows. Additionally, the enzyme's extracellular activity and the ease with which it can be monitored enhance its suitability for large-scale industrial applications, where efficiency and scalability are critical concerns.
Design Considerations
To optimize the expression of CelCD in E. coli, we applied several strategies. Codon optimization was performed to enhance translational efficiency in the host. We ensured that the GC content was within the optimal range for E. coli expression (40%), and illegal restriction sites were removed to comply with Biobrick standards.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 125
Illegal BglII site found at 261 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Protein Modeling
CelCD, was modeled with AlphaFold2 in Neurosnap. Overall, the model had high confidence which is likely due to the high sequence alignment with experimental structures as suggested by the sequence coverage plot. Further directions with this model may be to model the interactions with cellulose molecules. As this is an enzyme, modifications to its structure may be modeled to optimize enzymatic activity. Overall, the Ramachandran plot shows that the majority of the residues have acceptable phi and psi angles which verifies the confidence of the model.
Figure 2 | CelCD (Cellulase) predicted local distance difference test (pLDDT) plot generated by AlphaFold2.
Figure 3 | CelCD (Cellulase) multiple sequence alignment (MSA) sequence coverage plot generated by AlphaFold2.
Figure 4 | CelCD (Cellulase) model generated by AlphaFold2 and visualized with Jmol. GIF was generated by FirstGlance.
Figure 5 | CelCD Ramachandran plot generated by SWISS-MODEL.
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