Introduction

Our product, ArMOLDgeddon, aims to address mold growth on all kinds of indoor surfaces, including building walls, furnitures, ceilings, etc. Inspired by various stakeholders, we have also considered integrating our active ingredients into AC filters, creating an antimold filter material that rids our users off concerns about mold infestation in the ventilation system. To adapt our product to such a huge range of application scenarios, we have devised numerous product forms: (Fig. 1)

Fig. 1 | our product forms. For more information, please visit our ArMOLDgeddon page

However, we are faced with concerns that our enzymes may be easily washed away and may not consistently prevent mold growth at the targeted area. What's more, integrating enzymes into filter materials through commonly used methods – covalent bonding of enzymes to the polymers – may greatly affect enzyme activity. After discussion with professor Desen Ke, we have gained the concept of carbohydrate binding domain (CBM), and decided to fuse an additional CBM, namely BaCBM2 from Bacillus anthracis, to our main ingredients: chitinase and glucanase. Such addition confers our enzymes with high affinity to all kinds of polymer surfaces, lengthening the time period in which our enzymes stay in the targeted area, promoting long-term mold inhibition. Such CBM also allows our enzymes to bind to AC filter materials without affecting enzyme activity. Moreover, the CBM increases enzyme affinity to mold cell wall as well, promoting efficient mold deactivation. Therefore, we carried out investigation to characterize two novel BaCBM domains, CBM2 and CBM2e. CBM2e has an extra peptide sequence attached, which in theory, could increase the binding affinity of the domain to PET related materials [1] [2].

CBM2 & CBM2e
Method
To visualize the binding affinity of the two domains to carbohydrate materials, we expressed both proteins with GFP attached. Binding affinity assays are carried out on different materials, including gauze, PET film, rough and smooth sides of wallpaper, and writing paper. After sonic cell lysis, 1 μl of lysate supernatant is dropped onto each material. The system is then placed overnight in open environment to allow the solvent to completely evaporate. After overnight drying, the material is washed with distilled water for 10s, and the affinity is decided by visually inspecting the amount of solution left on the material. To investigate whether such addition would affect enzymatic activity, DNS assay is carried out on CBM-enzyme fusion body.
Results
Fusion proteins are successfully expressed with correct size, (Fig. 2) and affinity binding assay is carried out. After overnight drying, the materials are positioned under blue light for visual inspection. The results demonstrate that for all materials tested except writing paper, CBM2 possesses higher binding affinity to the material. (Fig. 3) Lack of difference between materials' binding ability on writing paper could be attributed to its innately rough and porous nature, making it a general anchor for any substance.
Fig. 2 | SDS-PAGE of two CBM domains fused with GFP, and CBM2e fused with BcChiA1 and Bglu1. s: supernatant of cell lysate; p: pellet of cell lysate
Fig. 3 | binding affinity assay for CBM2 and CBM2e on different materials: (a) rough side of wallpaper; (b)smooth side of wallpaper; (c) writing paper; (d) cellulose; (e) PET film
Three rounds of DNS assay are carried out for fusion proteins CBM2e-BcChiA1 and CBM2e-Bglu1. Results consistently demonstrate that addition of CBM domain does not decrease the activity of both chitinase and glucanase. (Fig. 4) Interestingly, addition of CBM domain to BcChiA1 increases its chitinolytic activity compared to its wildtype protein. This is possible due to increased affinity of the enzyme towards chitin molecules, conferred by the CBM domain.
Fig. 4 | DNS assay for fusion protein CBM2e-BcChiA1 on colloidal chitin and CBM2e-Bglu1 on lichenan.Error bars represent±SD (n=3).The data showed a statistically significant result in the t-test (p < 0.01)，indicated as **,(p < 0.0001)，indicated as ****.
Due to time constraints, fusion proteins CBM2-BcChiA1 and CBM2-Bglu1 have not reached successful expression, and DNS assay has not been carried out for these fusion proteins. However, since CBM2 and CBM2e are highly similar in terms of sequence and structure, CBM2 is expected to have the same effect on enzymatic activity of the two enzymes. We will further verify this in future experiments

Conclusion

Addition of CBM domains to our protein has demonstrated promising potential, with proved binding efficiency and little influence on enzyme activity. Such modification could greatly enhance the feasibility of many of our end products, paving the way for our commercial success.

Reference

  1. Rennison, Andrew Philip, et al. “Protein-plastic Interactions: The Driving Forces Behind the High Affinity of a Carbohydrate-binding Module for Polyethylene Terephthalate.” The Science of the Total Environment, vol. 870, Feb. 2023, p. 161948, doi:10.1016/j.scitotenv.2023.161948.
  2. Wang, Tao, et al. “Molecular Engineering of PETase for Efficient PET Biodegradation.” Ecotoxicology and Environmental Safety, vol. 280, June 2024, p. 116540, doi:10.1016/j.ecoenv.2024.116540.

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