Part:BBa_K5427002
KerDZ
Background Information
KerDZ is a versatile keratinase enzyme, notable for its unique ability to degrade a wide range of ester substrates, including BAEE, TAME, and BCEE. This functionality is crucial for breaking down the cuticle layers associated with keratins, such as those found in wool fabrics, enhancing the enzyme’s access to the substrate and improving degradation efficiency. In contrast to other bacterial keratin hydrolases that require the co-expression of multiple genes or operon systems, KerDZ operates as a single-component enzyme, effectively catalyzing keratin degradation without the need for additional enzymatic machinery.
Its broad applicability in industrial processes is supported by its high catalytic efficiency, stability under diverse conditions, and superior hydrolytic capacity. KerDZ functions effectively across a wide pH range (7-12) and temperature range (35-80°C), making it particularly well-suited for thermo-alkaline environments. Comparative studies indicate that KerDZ outperforms other keratinolytic proteases, such as KerAK-29, as well as widely-used commercial enzymes like subtilisin Carlsberg, in terms of catalytic efficiency, hydrolysis rate, and stability in the presence of polyols. Moreover, KerDZ demonstrates excellent compatibility with a variety of commercial detergents, positioning it as a cost-effective and environmentally sustainable alternative to the toxic chemicals traditionally used in leather processing and detergent formulation. These features highlight KerDZ's potential for further development in various industrial applications, including biotextiles and the detergent industry.
Design Considerations
To optimize the expression of KerDZ in E. coli, we applied several strategies. Codon optimization was performed to enhance translational efficiency in the host. Repetitive sequences were manually removed to prevent issues with genetic instability. We ensured that the GC content was within the optimal range for E. coli expression (37%), and illegal restriction sites were removed to comply with Biobrick standards. Alongside this KerDZ has two domains: a pro-protein and the proteolytic domain. We considered keeping the pro-protein within our clones as it is involved in the in vivo folding of the proteolytic domain and is naturally cleaved following secretion. If we only cloned the proteolytic domain this had the potential of affecting the in vivo folding of the enzyme, resulting in a loss of function.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Part Assembly
We constructed a synthetic plasmid to express the keratin hydrolase, KerDZ. The KerDZ gene, originally derived from Actinomadura viridilutea strain DZ50, includes a tandem polyhistidine (His) tag at the C-terminus of the gene and was synthesized and ordered from IDT. The pJUMP28-J23119-RBS1 vector backbone underwent sequential double digestion using the restriction enzymes SpeI and PstI. To introduce compatible restriction sites for pJUMP28, the KerDZ gene block was amplified via PCR using the forward primer P_pre10 (iGEM 2023 Designation #BBa_K4755025) and the reverse primer P_suf10 (iGEM 2023 Designation #BBa_K4755027). Both primers were sourced from the UAlberta iGEM 2023 project’s primer collection. Successful amplification was confirmed by agarose gel electrophoresis. The PCR product was subsequently double-digested with XbaI and PstI and ligated into the vector using NEB T4 DNA Ligase according to standard protocols.
Characterization of KerDZ
Once our construct was created with our keratin degradation enzyme, we transformed this plasmid into DH5alpha, BL21, and Rosetta Gami, and performed a growth curve analysis. As seen in in previous phases of our project, salt stress may have a small effect on the cell metabolism, therefore we grew each strain in regular and low salt LB. Cultures were grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm.
The growth curves of each E. coli strain in regular LB and low salt LB categorized the metabolic effect of salt between each strain, as well as highlighting the overall growth rate. Figure 1 showed the effects of each strain of E. coli when growing in regular LB which allowed us to examine the effect our plasmid had on each strain. We then concluded that our KerDZ construct grows most effectively in DH5alpha and BL21, notably at 10 hours we saw a 51.85% and 23.67% increase in growth, respectively, in comparison to Rosetta Gami at the same time mark. Although we observed adequate growth from both BL21 and DH5alpha, when compared to the empty vector controls DH5alpha under performed, whereas BL21 grew more than its control. These results are consistent with what we observed previously where BL21 grew better than its control and DH5alpha the opposite. Therefore we determined that either BL21 or DH5alpha would be suited for transformation of our vector. We did not exhibit any significantly large inhibitions of growth between any strain. We then performed this experiment again with cells incubating in Lennox LB (low sodium). Figure 2 indicates the same growth factors as seen in figure 1. Even in low salt concentrations KerDZ grew the most in DH5alpha, and then BL21, where Rosetta Gami performed the worst. Consistent with our previous growth curve, BL21 grew better than its empty vector control, and DH5alpha grew less than its control. These tests confirm previous results and allow us to conclude that either BL21 or DH5alpha would give us adequate growth with our constructs. We then compared figure 1 and figure 2 to determine if there was any significant difference in salt concentrations on growth. Comparing the OD measurements at 10 hours for each strain between low salt conditions and high, we determined that these cells grew better in regular LB. Rosetta Gami at 10 hours of growth had an average OD measurement of 0.38 in regular LB but a measurement of 0.269 in Lennox LB, meaning we saw a 29% increase in growth for regular LB. Similarly, BL21 showed a 16.6% increase in growth in regular LB at the 10th hour. Contradictorily, Rosetta Gami when comparing both regular and Lennox LB saw a 0.25% reduction in growth in the regular LB at hour 10. These results indicate that there is very little if any salt stress happening in the cells for regular LB compared to Lennox. We then compared this result to our phase 3, figure 2 growth curve which tests a similar construct but without KerDZ, where we saw an increase in growth during the low salt condition. Since in figure 2 we saw the opposite results, where lower salt inhibited the growth of our organism, we determined that the KerDZ construct must have a higher tolerance to salt concentrations within the media, which allowed the plasmids to grow better in higher salt than seen in previous experiments. We can also conclude that there is very little effect that salt concentration has on the overall growth of the E. coli strains, and there was no real significant difference between the variables themselves.
Figure 1 | Growth Curve for J23119_RBS 1_KerDZ_pJUMP 28 in DH5a, R. Gami and BL21 at 37 degrees in Regular LB. Cultures were grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm.
Figure 2 | Growth Curve for J23119_RBS 1_KerDZ_pJUMP 28 in DH5a, R. Gami and BL21 at 37 degrees in Lennox LB Cultures were grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm.
Protein Modeling
As suggested by Elhoul et al. in 2016, KERDZ has two different domains. The first is the pro-protein domain which is involved in the proper in vivo folding of the protein. This domain is cleaved upon the protein’s secretion by other proteases. As such, this domain was not modeled as it does not complete the cleaving of keratin. The other domain was the protease domain containing the catalytic site involved in keratin degradation. This domain was modeled separately based on the amino acid region stated by Elhoul et al. in 2016 and used as a standalone to better represent the interactions between KerDZ and keratin within in vitro assays or natural keratin degradation. This model had high confidence for all of the residues which is due to the alignment with experimentally derived protein structures as shown by the sequence coverage. The Ramachandran plot also shows that nearly all of the residues have acceptable phi and psi angles.
Figure 3 | Domains of KerDZ derived from NCBI’s Conserved Domain Search.
Figure 4 | KerDZ (Keratinase) predicted local distance difference test (pLDDT) plot generated by AlphaFold2.
Figure 5 | KerDZ (Keratinase) multiple sequence alignment (MSA) sequence coverage plot generated by AlphaFold2.
Figure 6 | KerDZ (Keratinase) model generated by AlphaFold2 and visualized with Jmol. GIF was generated by FirstGlance.
Figure 7 | KerDZ Ramachandran plot generated by SWISS-MODEL.
KerDZ and Keratin Interaction
An HDOCK model was done for KerDZ and keratin. The results also suggested that there are interactions that can occur between KerDZ and keratin. The docking scores are around -200, suggesting the interaction is likely. Furthermore, a confidence above 0.7 suggests a likely interaction (Yan et al., 2020). The ligand rmsd is not a measure of the docking accuracy and is only used to show the difference between the 3D structure of the model and the input structures. This models only one of the many interactions with natural wool proteins. Modeling other interactions of natural keratin proteins with KerDZ may be done.
Figure 8 | KerDZ and Keratin31 interaction (Rank 1) generated by HDOCK. Image was generated with SWISS-MODEL.
Figure 9 | KerDZ (Keratinase) and Keratin31 interaction model (Rank 1) generated by HDOCK and visualized with Jmol. GIF was generated by FirstGlance.
Table 1 | HDOCK output containing docking score, confidence score, and the ligand rmsd of the top 10 protein-protein interactions for KerDZ and Keratin31.
KerDZ and Biosensor Interaction
Preliminary modeling for the interaction between the keratinase (KerDZ) and the biosensor (Q-sensor:BBa_K5427077, but instead of nanobody insertion domain, containing keratin 31) to visualize how KerDZ would interact with our synthetic construct was also completed using HDOCK.5, 11, 12, 13 To do this, the highest quality models (highest ranking model) for the protease domain of KerDZ and the biosensor were used as the inputs. HDOCK completes protein docking and generates several models of the highest likely interactions between the two chains. The output provides a 3D model that highlights the domains where the interaction occurs. It also yields a docking score and confidence score. These results showed that there is high confidence (> 0.90) that the KerDZ interacts with the keratin 31 portion of our synthetic construct, implying proper catalytic activity. This also has implications for the interaction with natural keratin 31 found in textiles. Docking scores below -200 are also indicative of a good model. The docking scores all are < -200 for the top 10 models. The ligand rmsd is not a measure of the docking accuracy and is only used to show the difference between the 3D structure of the model and the input structures.
Figure 9 | KerDZ (Keratinase) and Biosensor interaction model (Rank 1) generated by HDOCK and visualized with Jmol. GIF was generated by FirstGlance.
Table 2 | HDOCK output containing docking score, confidence score, and the ligand rmsd of the top 10 protein-protein interactions for KerDZ and biosensor (containing keratin 31).
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