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TPA ENZYME MIX

Enzymatically converting TPA to PCA for detection

The enzymes needed to convert TPA to PCA are available in the registry from Darmstadt 2012. By the time PcaU was syntesized, we did not have enough time to use Darmstadt’s parts which required assembly. We contacted professor Eiji Masai, who generously provided us with the necessary plasmids to express the enzymes in E. coli. These are pETtpa23, pRSFtpa1 and pETtpb from Masai’s 2006 paper.

Figure from Masai et. al.
The terepthalic acid degradation pathway. It is an aerobic process that requires the presence of NADPH, although the NADPH is restored at the end of the proccess.

The plasmids were expressed in BL21 DE3 E. coli and lysate was prepared using the protocol below.

Expression was verified using the same SDS-PAGE protocol employed by Darmstadt 2012, for which we obtained similar results.

TPH SDS Gels
SDS gels of TPH enzymes in E. coli whole cell fraction from the protocol shown earlier. Lanes are denoted by letter code and hours of incubation post induction. For example, B4h = TphB 4 hours post induction. Blue boxes denote bands at molecular weight matching the Darmstad's result. The purple box is an unexpected, unknown band. Severe leaky expression of TphA1A2 and TphB can be seen in the zero hour fractions. However, our results in the demonstration section show that this has not affected the enzyme's function.

We tested TPH enzyme functionality in various buffers to develop a final buffer, TPH buffer. TPH buffer is a buffer that must maintain TPH enzyme function in the presence of PETase buffer. This is because the resuspended TPH enzymes will be applied to supernatant from PETase degradation containing TPA. The TPH enzymes will convert TPA to PCA for detection by PcaU. Therefore, these buffers must also not prevent growth of PcaU transformed E. coli cells.

10mM Tris buffer at pH 7.2 was chosen for PETase buffer and TPH buffer. PNPB tests from multiple teams including our own have shown that PETase is functional in Tris, and our own growth tests revealed that although Tris may be somewhat toxic, PcaU transformed BL21 E. coli are able to grow in the presence of 10mM tris buffer. TPH buffer also contains some key components used in Masai’s work, including 0.1mM ferrous ammonium sulfate (required iron supply), 2mM L-Cysteine Hydrochloride, and 10% glycerol (for preservation). We also added 100um NADPH to this buffer to speed the reaction

PETase has worked in PBS, but TPH enzymes may not due to NADPH being unstable in phosphate buffers. PBS also precipitated when ferrous ammonium sulfate was added to it, likely due to formation of insoluble ferrous ammonium phosphate. PBS is not a suitable buffer for TPH enzymes

sources

1. Sasoh, M., Masai, E., Ishibashi, S., Hara, H., Kamimura, N., Miyauchi, K., & Fukuda, M. (2006). Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6. Applied and Environmental Microbiology, 72(3), 1825–1832.

2. Schläfli, H. R., Weiss, M. A., Leisinger, T., & Cook, A. M. (1994). Terephthalate 1,2-dioxygenase system from Comamonas testosteroni T-2: purification and some properties of the oxygenase component. Journal of Bacteriology, 176(21), 6644–6652.

3. Yuki Fukuhara, Daisuke Kasai, Yoshihiro Katayama, Masao Fukuda & Eiji Masai (2008) Enzymatic Properties of Terephthalate 1,2-Dioxygenase of Comamonas sp. Strain E6, Bioscience, Biotechnology, and Biochemistry, 72:9,2335-2341, DOI: 10.1271/bbb.80236