Part:BBa_K3759019
mLCC-linker-BsLA
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079
Illegal NheI site found at 193 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079 - 1000COMPATIBLE WITH RFC[1000]
Usage
It has been well known that the surface of PET film is hydrophobic, and the surface of mLCC is hydrophilic. By constructing the mLCC-linker-BsLA fusion protein, the PET degradation efficiency will be enhanced enormously, due to the unique properties of amphiphilicity and self-assembly of hydrophobin BslA. Also, as BslA was extracted from bacteria and was a bacterial hydrophobin, it shows a better fusion with mLCC, which help the increment of the PET degradation efficiency of mLCC-linker-BslA.
Biology
LCC is a leaf-branch compost cutinase[1] and a kinetically robust protein[2]. A research published on Nature came up with a mutant enzyme, mLCC[1] 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 the monomers found in petrochemical materials.
The linker is GGGGSGGGGS.
BslA is a structurally defined bacterial hydrophobin that was found in the biofilm of Bacillus subtilis. It helps the assembling of TasA (an exopolysaccharide and an amyloid fiber-forming protein), the component of the biofilm matrix. BslA is composed of an Ig-type fold with the addition of an unusual, extremely hydrophobic “cap” region. The central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. [3]
Design Consideration
The construct was cloned into a pET28a plasmid and transformed into BL21 (DE3) E. coli.
The construction includes:
1. a 6× His tag is added to enable us carrying out Ni-NTA protein purification.
Protein Expression
Figure 1. The expression of mLCC-linker-BslA (Left 1st 2nd)
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 24 hours. Then, we used 200mM imidazole to eluting and get left 1st aimed protein, and we used 300mM imidazole to eluting the left 2nd aimed protein.
4. PET Degradation Reaction
We perform PET degradation reaction to detect enzyme activity. We set several protein concentration to detect enzyme activity, under the condition of pH8,70°C, and 18h of reaction time. Then we measured the absorption value at uv240nm by nanodrop, which is the absorption position of the product TPA, and we were surprised to find that the relative enzyme activity of mLCC-linker-BslA was increased about 3 times compared to mLCC!
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">Fig. (a). The adsorption value of mLCC, mLCC-Linker-BslA of UV240 under the condition of pH8, 70 celsius degree, reaction time of 18h.
(b) The relativity activity of mLCC, mLCC-Linker-BslA.
References
[1] 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.
[2] Sulaiman S , You D J , Kanaya E , et al. Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase[J]. Biochemistry, 2014, 53(11):1858-1869.
[3]: “BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm.” Proceedings of the National Academy of Sciences of the United States of America vol. 110,33 (2013): 13600-5. doi:10.1073/pnas.1306390110
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