Part:BBa_K4290018

pET22b-PelB-SbPETase

Profile

Name: pET22b-PelB-SbPETase

Base Pairs: 6219 bp

Origin: Escherichia coli, synthtic

Properties: pelB is fused with SbPETase to achieve SbPETase secretion

Usage and Biology

Bba_K4290018 is the construct of SbPETas, with the N-terminal is fused with the pelB. The pectate lyase B signal peptide (PelB), is a Sec-dependent translocation signal peptide for directing the target protein to the bacterial periplasm [1]. SbPETase is a novel enzyme for Poly (ethylene terephthalate) (PET) hydrolysis. PET is one of the most commonly used plastics worldwide and its accumulation in the environment is a global problem. PETase has been reported to exhibit higher hydrolytic activity and specificity for PET than other enzymes at ambient temperature [2]. Enzymatic degradation of PET using PETase provides an attractive approach for plastic degradation and recycling [3]. Various enzymes showing PET depolymerization activity have been identified and investigated in recent years, such as cutinases, PETase, MHETase, lipases, and esterases[4-9]. SbPETase can degrade PET into small fragments, then transport the degraded products for further "digestion", and finally convert them into two relatively simple organic compounds, ethylene glycol, and terephthalic acid [3].

Figure 1. Schematic extracellular production of SbPETase in Escherichia coli..

Construct design

1. pET22b-PelB-SbPETase construction

The pelB was fused with the N-terminal of SbPETase and ligated it to the double-enzyme digestion pET22b vector for the construction of (Figure 2).

Figure 2. Schematic map of pET22b-PelB-SbPETase.

The profiles of every basic part are as follows:

BBa_K4290021

Name: pelB

Base Pairs: 147 bp

Origin:pET22b, plasmid

Properties: a Sec-dependent translocation signal peptide

Usage and Biology

BBa_K4290021 is a coding sequence of pelB, the Sec-dependent translocation signal peptide for extracellularly expression in the Escherichia coli.

BBa_K4290022

Name: sbPETase

Base Pairs: 66 bp

Origin: S. brevitalea sp. nov, genome

Properties: a novel enzyme for Poly (ethylene terephthalate) (PET) hydrolysis.

Usage and Biology

BBa_K4290022 is the coding sequence of SbPETase.

Experimental approach

1. Electrophoresis of the constructed pET22b-PelB-SbPETase

The amplify SbPETase from the genome of S. brevitalea sp. nov (Figure 3A.) was ligated to the double-enzyme digestion pET22b vector. The recombinant plasmids pET22b-PelB-Sbpetase was 6240 bp in length. To verify if the plasmid is correct, plasmid pET22b-PelB-SbPETase was digested with ApaLI (Figure 3C). We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 3D), and the plasmids were successfully constructed. So far, we have successfully obtained the recombinant plasmids.

Figure 3. Gel electrophoresis and sequening of pET22b-PelB-SbPETase.A. The gene fragment of SbPETase; B. digest plasmid pET22b-PelB-SbPETase with ApaLI; C. the sequencing result of the recombinant plasmid pET22b-PelB-SbPETase..

2. Protein expression and purification

Figure 4. The expression assessment of SbPETase.A, SDS-PAGE result of SbPETase; B. Yield compariosn of ertracellular and intracellular SbPETase...

We transferred the plasmid pET22b-pelB-SbPETase into E. coli BL21(DE3), expanded the culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column (Ni-NTA) purification to purify the protein SbPETase (Figure 4A). In order to test the extracelluar presence of SbPETase protein, we compared the extracellular and intracellular SbPETase (Figure 4B). In Figure 4B, the extracellular SbPETase was observed.

3 Biochemical characterization of SbPETase

We set up an in vitro system and confirmed the ability to use the SbPETase to degrade PET materials. As shown in Figure 5, we demonstrated that SbPETase could degrade PET and BHET into MHET and a small quantity of TPA through HPLC (Figure 5A, B), the optimum conditions showed that at 30°C, pH 7.0 (BHET as substrate) or pH 8.0 (PET film as substrate), SbPETase showed the highest activity.

Figure 5. Two different HPLC experiments of SbPETase degrade PET and BHET materials. A. result recorded with an HPLC system successfully identified the MHET and BHET peak when used BHET as substrate, B. result recorded with an HPLC system successfully identified the MHET, BHET,and TPA peak when used PET as substrate..

References

1.Kusuma S. A. F et al. (2022). Improving of pelB-Secreted MPT64 protein released by Escherichia coli BL21 (DE3) using Triton X-100 and Tween-80. J Adv Pharm Technol Res, 13(3):171-176.

2.Sinha, V.; Patel, M. R.; Patel, J. V. PET waste management by chemical recycling: a review. J. Polym. Environ. 2010, 18, 8−25.

3.Shi L, Liu H, Gao S, et al. Enhanced extracellular production of is PETase in Escherichia coli via engineering of the pelB signal peptide[J]. Journal of Agricultural and Food Chemistry, 2021, 69(7): 2245-2252.

4.Kawai, F.; Kawabata, T.; Oda, M. Current state and perspectives related to the polyethylene terephthalate hydrolases available for biorecycling. ACS Sustainable Chem. Eng. 2020, 8, 8894−8908.

5. Ronkvist, Å. M.; Xie, W.; Lu, W.; Gross, R. A. Cutinase catalyzed hydrolysis of poly(ethylene terephthalate). Macromolecules. 2009, 42, 5128−5138.

6. Tournier, V.; Topham, C. M.; Gilles, A.; David, B.; Folgoas, C.; Moya-Leclair, E.; Kamionka, E.; Desrousseaux, M. L.; Texier, H.; Gavalda, S.; Cot, M.; Guémard, E.; Dalibey, M.; Nomme, J.; Cioci, G.; Barbe, S.; Chateau, M.; André, I.; Duquesne, S.; Marty, A. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 2020, 580, 216−219.

7. Barth, M.; Honak, A.; Oeser, T.; Wei, R.; Belisario-Ferrari, M. R.; Then, J.; Schmidt, J.; Zimmermann, W. A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films. Biotechnol. J. 2016, 11, 1082−1087.

8. Nikolaivits, E.; Kanelli, M.; Dimarogona, M.; Topakas, E. A middle-aged enzyme still in its prime: recent advances in the field of cutinases. Catalysts 2018, 8, 612.

9. Yoshida, S.; Hiraga, K.; Takehana, T.; Taniguchi, I.; Yamaji, H.; Maeda, Y.; Toyohara, K.; Miyamoto, K.; Kimura, Y.; Oda, K. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 2016, 351, 1196−1199

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 1276
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 1276
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 5233
Illegal XhoI site found at 6022
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 1276
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 1276
Illegal NgoMIV site found at 273
Illegal NgoMIV site found at 3453
Illegal NgoMIV site found at 3613
Illegal NgoMIV site found at 5201
Illegal NgoMIV site found at 5394
1000
COMPATIBLE WITH RFC[1000]