Difference between revisions of "Part:BBa K1921000"

Latest revision as of 15:31, 21 October 2016

PETase

Sequence and Features

Usage

PETase is dedicated to the role of PET degradation. Our subject of the competition for this year is “training the molecule for the environment”,and the PETase is our training object.Our training methods act in three following aspects.
Firstly, we find the enzyme catalytic center and its binding center by analyzing the structure of PETase with Protein crystallography and X ray diffraction technique, as well as choosing the mutation site under its character direction in order to carry out the directional mutation to improve the degradation efficiency and thermal stability.
Moreover, using the prokaryotic (E. coli) and eukaryotic (Pichia Pastoris) surface display for whole cell catalysis.
Thirdly, fusing the PETase and hydrophobic protein then expressing the fusion protein in Pichia Pastoris, which will take advantage of hydrophobic protein in hydrophobicity to give a hydrophobic environment for a better degradation efficiency. Meanwhile, the co-display combining PETase and hydrophobic protein in Pichia Pastoris will change the character of cells' surfaces so that cells can adapt the extreme environment, then the whole-cell biocatalyst might have higher catalytic efficiency as well as break the limitation set by reaction condition. According to that, the PETase degradation reaction conditions will be broaden which means it can be applied in industry easier.

Biology

PETase was found from a kind of microorganism（Ideonella sakaiensis 201-F6） living on PET as the main carbon source. It can degrade macromolecular polymers into monomers.PETase is the only enzyme found in bacteria which can degrade PET. Compare to the other enzyme found in fungi like LCC, TfH, FsC, PETase is much more active under low temperature environment, which means its reaction conditions is feasible in practical application than the others'.Additionally, PETase has been shown to have a degrading efficiency 120 times greater than alternative enzymes.

Reference

[1] Yoshida S, Hiraga K, Takehana T, et al. A bacterium that degrades and assimilates poly(ethylene terephthalate).[J]. Science, 2016, 351(6278):1196-1199.

Structure

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with ampicillin.

Cultured in bottles:
After 4 hours culturing in 37℃ in bottles, we used 500μM IPTG induced in 16℃ for 8-12h.

Ni-sepharose purification:
The supernatant was applied to the His-Accept nickel column. After washing unbound proteins with the lysis buffer(50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 20 mM imidazole), the bound proteins were eluted with elution buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 250 mM imidazole).

Ultrafiltration:
To reduce the salt concentration, we use evaporating pipe to reduce the liquid volume to 1/6 and then add 5/6 A liquid. By using ultrafiltration at the speed of 3500rpm, we finally get 5mL protein solution.

Cation exchange column:
Use Hitrap SP HP 5mL column to go through AKTA system to get the protein with certain pI.

Ultrafiltration:
Use the evaporating pipe to concentrate the solution to 0.5mL.

Gel filtration chromatography:
Use AKTA system and superdex75 gel filtration chromatography to separate proteins with different molecular weight.

Crystalization: Using the way of vapor diffusion and sitting drop to grow good crystal to do X Ray diffraction.

HPLC Result

Figure 1. PETase’s self-degrading condition in different temperature. We take the quantity of PETase in the day the experiment begines as 100%. We can now see when stored in low temperature, the protein was degraded slowly, but in room temperature, the protein degrades rapidly. The Quantity of PETase left was measured by protein gel and analysised by a computer program called GEL-PRO.

Mutation

Figure 2. The result of the purification of PETase and its 3 mutants. The 4 kinds of protein are purified trough nickel columns.

Figure 3. The result of pre-expression of pET-21b-MutateD/J/M and pET-21b-PETase.“+” is induced with IPTG,“-” is not induced with IPTG.

Figure 4. The Comparison of the enzyme activity between PETase and three kinds of mutated PETase. The reaction condition is 100μL solution,pH 9.0(bicine-NaOH), 40 degree, 18h, the substrate is a round with a diameter of 2mm. The results are detected by Hplc. The y-axis stands for the area of the peak of MHET, the main product of the PETase’s degrading of PET. The x-axis stands for the concentration of the protein.

Surface display in E.coli by using INP

Figure 5. Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 16℃.

Figure 6. Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 25℃ with different amount of bacteria.

Figure 7. Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced with 0.1mM IPTG for 24h.

Figure 8. Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)NP when induced at 16℃ with 0.1mM IPTG for 1h, 4h, 8h, 12h, 16h and 20h.

Surface display in E.coli by using LPP-OmpA

Figure 9. Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)LAP when induced at 16℃.

Surface display in E.coli by using BrkA

Figure 10. Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)Brk when induced at 16℃ and 25 ℃ with 0.02mM IPTG.And the last two were induced with 0.09mM IPTG.

Surface display in E.coli by using AIDA

Figure 11. Reletive enzyme activity of engineering  bacteria E.coli(BL21)/pET22b(+) ap at 16 ℃.

Surface display in Pichia Pastoris

<p style="text-align: center;">
Figure 12. The activity of P. pastoris PETase-GCW21. a&b used the first group of yeast; c&d used the second of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.

<p style="text-align: center;">
Figure 13. The activity of P. pastoris PETase-GCW51. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.

<p style="text-align: center;">
Figure 14. The activity of P. pastoris PETase-GCW61. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.

Co-display in Pichia Pastoris

Figure 15. The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 16. The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 17. The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 18. The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours and amount of yeast.

Figure 19. The activity of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformant and ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformant in best condition.