Difference between revisions of "Part:BBa K3715005"

Revision as of 14:31, 13 October 2022

Super5

PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbeIdeonella sakaiensis. Well, Super5 is a complicated mutation of PETase, which contains 11 mutation sites：S214H-I168R-W159H-S188Q-R280A-A180I-G165A-Q119Y-L117F-T140D-S121E.

Sequence and Features

Origin(organism)

Ideonella sakaiensis

Molecular cloning

First, we used the vector pET21a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

Figure 1. The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(50μg/mL) in 37℃ overnight.
Massive expressing:
After taking samples, we transfered them into 900ml LB medium and added antibiotic to 50 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 5-6 hours). Induce the culture to express protein by adding 0.5 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.

Affinity Chromatography:
We used the Ni Agarose to purify the target protein. The Ni Agarose can combine specifically with the Ni-His tag fused with target protein.

• First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.
  
• Second, add the protein solution to the column, let it flow naturally and bind to the column.
  
• Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  
• Forth,add Ni-Washing buffer several times. Check as above. Collect the eluted proteins for further operation.
  

Figure 2. The result of SDS-PAGE.

Gel filtration chromatography:
The collected protein samples are concentrated in a 30 KD concentrating tube at a speed of 3400 rpm and concentrated for a certain time until the sample volume is 500 μl. At the same time, the superdex 75 column is equilibrated with a buffer to balance 1.2 column volumes. The sample is then loaded and 1.5 cylinders are eluted isocratically with buffer. Determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.

Figure 3. The result of gel filtration used the superdex75 column with the AKTA system.

Enzyme activity determination

We use HPLC equipment to measure the peak area of the product of PET(MHET) of the reaction, in order to express the enzyme activity of PETase. For more information on the product of PET(MHET), please see our project introduction.

Figure 4. Enzyme activity determination, compared with wild type.

Conclusion

In conclusion,the enzyme activity and thermostability of Super5 has greatly improved 163 times ，compared with WT(wild type).

Improvements

Group: BJEA_China 2021

Author:Chenzhang Ma

Summary: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.

BslA is the only bacterial hydrophobin identified so far. It is similar in properties to fungal hydrophobins.

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 2022, the TJUSLS_China discovered a mutant super5, which is mPETase (BBa_K3715005). The degradation efficiency of mPETase is 163 times that of wild type, which greatly improves the degradation efficiency of PET. Therefore, this year we decided to further improve mPETase with excellent degradation efficiency. Our team is devoted to increasing PET degradation efficiency and improving mPETase. Therefore, we proposed the construction of a fusion protein of mPETase and BslA, hoping to improve the PET degradation efficiency by improving the adsorption capacity of mPETase enzyme on the hydrophobic PET film.

By constructing the mPETase-GSlinker-BslA and mPETase, 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.

Molecular Cloning

For molecular cloning, we selected pET28a as vector. We successfully amplified two gene segments of mPETase (as control group), mPETase-GSlinker-BslA (Figure 1a). Then we digested and connected all the segments to pET28a vector through two restriction enzymes of BamHI and XhoI. At present, two recombinant plasmids have been successfully constructed (Figure 1b).

Figure 1. (a) PCR results. M: marker 1-6: mPETase(795bp) 7: mPETase-GSlinker-BslA(1242bp)

(b) Enzyme digestion verification results. M: marker 1: mPETase(795bp) 2-4: mPETase-GSlinker-BslA(1242bp) ‍

Reference

[1] Puspitasari, Nathania, Shen-Long Tsai, and Cheng-Kang Lee. "Fungal hydrophobin RolA enhanced PETase hydrolysis of polyethylene terephthalate." Applied Biochemistry and Biotechnology 193.5 (2022): 1284-1295.

[2] Ribitsch, Doris, et al. "Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins." Applied and Environmental Microbiology 81.11 (2015): 3586-3592.

GreatBay_SCIE 2022's Characterisation

Dockerin-fused variant of Super-5-mut PETase

PETase5-dockerin is an improved version of Super-5-mut PET hydrolase from the iGEM team TJUSLS_China (Part: BBa_K3715005). This high-efficiency, thermostable, durable super mutant consists of 11 mutation sites compared to the wild-type: S214H, I168R, W159H, S188Q, R280A, A180I, G165A, Q119Y, L117F, T140D, S121E [1]. The improvement is implemented by fusing the original sequence design with a dockerin I domain at the C' terminal to allow its high-affinity anchorage onto the CipA scaffoldin and the rest of the polyester degradation complex. The catalytic domain of PETase5-t and the dockerin domain are interspaced with a medium-lengthed flexible GS linker (10 aa long) to avoid steric inhibitions.

The artificially-designed PETase5-Dockerin I fusion protein could be tightly-anchored onto the CipA scaffoldin via the high-affinity Doc I: Coh I noncovalent interaction. The CipA primary scaffoldin is then tightly-anchored onto the secondary scaffoldin - OlpB, which is either anchored onto the cell surface of K.marxianus via ScGPI, or binds to E.coli's Cell-surface Nanobody3(Nb3)(BBa_K4275026). It is believed that the immobilization of the two enzymes (PETase5-dockerin and MHETase-t(BBa_K4275010)) could increase their proximity and further enhance their synergy, whilst the affinity of carbohydrate-binding module 3 (CBM3) on the CipA scaffoldin towards PET fiber could further increase the catalytic efficiency of this degradation complex.

Figure 2: Fig.2 PETase-5 expression (A) Metabolic pathway of PET degradation, PETase catalyzes the cleavage of PET into MHET (mono-2-hydroxyethyl terephthalate) and EG (Ethylene glycol). (B) Genetic circuit constructions of PETase-5 and PETase5-Dockerin with type I dockerin fused to anchor the enzyme subunit onto the cellulosome complex. (C) SDS-page analysis for PETase 5 and PETase 5-Dockerin. (D) The PH values of different samples of PET degraded by PETases either fused or not fused with type I dockerin domain.