Hydrophobins are small surface-active proteins, and have both fungal and bacterial origins. Hydrophobins originated from fungi are divided in to two class, and are being widely used and applied. HFBI[1] and HGFI[2] are hydrophobins derived from fungi, HGFI is a class Ihydrophobin, and HFBI is a class II hydrophobin. Hydrophobin’s hydrophobic part is exposed on the surface forming a planar area called the hydrophobic patch, making the surface of the protein contain both hydrophobic and hydrophilic area therefore making the surface making the hydrophobins amphiphilic. Because of the hydrophobins’ amphiphilicity property it can self-assemble themselves with others. In 2015, the teamTianjin mutated these two hydrophobins, and the resulting mHGFI (in Janus-m, http://parts.igem.org/Part:BBa_K1582002 ) and mHFBI (sJanus-m，http://parts.igem.org/Part:BBa_K1582003) can be expressed in bacteria.
Recently, are search published on Nature came up with a mutant enzyme, mLCC[3]that hydrolyzes 90% of PET in plastic bottles in just 10 hours. This is more efficient than any previous PET hydrolase, and more importantly, the resulting monomers- ethylene glycol and terephthalic acid have the same properties as themonomers found in petrochemical materials.
Our team is devoted to increasing the activity of mLCC and reducing enzyme purification costs. Hydropobins fusion with mLCC is an efficient method which increasing the efficiency of degradation by enhancing cell surface adsorption, since the surface of PET film is hydrophobic and the surface of mLCC is hydrophilic. By constructing the mLCC-linker-mHFBI, mLCC-linker-mHGFI and mLCC-linker-BsLA fusion protein, the PET degradation efficiency will be enhanced due to the unique properties of amphiphilicity and self-assembly of hydrophobins.
To sum up the above, we use our practice to make some contribution to PET degradation in order to protect the environment.
Pre-expression:
The BL21 bacteria that contains aimed protein were cultured in 5mL LB liquid medium with kanamycin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with kanamycin.
Cultured in bottles:
After culturing in 37℃ in bottles, we used 0.5mM IPTG induced in 16℃ for 24hours. Then, we used 200mM imidazole to eluting and get left 1st, 3rd, 5th, 7th aimed protein, and we used 300mM imidazole to eluting the left 2nd, 4th, 6th, 8th aimed protein.
[1] J Vereman, Thysens T , Derdelinckx G , et al. Extraction and spray drying of Class Ⅱ hydrophobin HFBI produced by Trichoderma reesei[J]. ProcessBiochemistry, 2019, 77(FEB.):159-163.
[2] Song D , Wang X , Gao Z , et al.Expression, Purification and Characterization of Hydrophobin HGFI from GrifolaFrondosa in Saccharomyces Cerevisiae[J]. Acta Scientiarum NaturaliumUniversitatis Nankaiensis, 2018.
[3] Tournier, V., Topham, C. M. , Gilles, A. , David, B. , & Marty, A. .(2020). An engineered pet depolymerase to break down and recycle plastic bottles. Nature, 580(7802), 216-219.
