Difference between revisions of "Part:BBa K3898165"

Revision as of 00:17, 22 October 2021

RIAD-hydrophobin4

e

Sequence and Features

RIAD-hydrophobin4 consists of two components RIAD (BBa_K3898000) and hydrophobin4 (BBa_K3898002). This is a part of our PET degradation three-enzyme complex . Hydrophobin4 are involved in our designed system with possible functions of adhering to PET polymers and altering the physicochemical properties of PET for degradation improvement

PET plastic sheet degradation test

We obtained PET plastic sheet with 12% crystallinity (scientific research only) from TJUSLS_China, and cut it into 5mm*5mm fragments (0.07g per fragment). We incubated 1.5 mL of E.coli BL21, E.coli BL21/pET28a-PD, E.coli BL21/pET28a-M, E.coli BL21/pET28a-PD-MD-hA cell pellet and medium in several EP tubes，and added three 5mm*5mm fragments, respectively. We then incubated the reaction mixture at 37℃ for 7 days. After 7 days, the degradation product, erephthalic acid (TPA), was detected by UV Spectrophotometry and thus determined PET degradation efficiency.
In terms of measurement, we chose to use UV spectrophotometry to detect the output of TPA. Binding with RIDD-PETase and RIDD-MHETase, PET will be decomposed with ethylene glycol (EG) and terephthalic acid (TPA) as final product. Ethylene glycol is volatile, and the test results are not credible, so we decided to detect TPA. Through previous literature research, we found that there are two mainstream detection methods for TPA. One is to directly perform UV spectrophotometry on the sample. The increase in absorbance of the reaction mixture in the ultraviolet region of the light spectrum (at 240 nm) indicates the release of soluble TPA or its esters from an insoluble PET substrate. This compound shares an identical strong absorbance peak around 240–244 nm with an identical extinction coefficient as all three compounds contain the same number of carbonyl groups. The second is to adopt reverse-phase HPLC. Reverse-phase HPLC systems have been widely used to analyze the products derived from the enzymatic hydrolysis of PET owing to their powerful resolving capability and reproducibility. The different compounds produced by PET hydrolytic enzymes (i.e., TPA, MHET, and BHET) can be efficiently separated on a C18 reverse-phase HPLC column: The reaction mixture is loaded into a column equilibrated with a polar mobile phase and the concentration of the organic solvent (acetonitrile).
Considering our experimental cycle, throughput, and laboratory conditions, we chose UV spectrophotometry to detect TPA. Then we made the standard curve of TPA at OD240 (Figure12)
. The liquid obtained after incubation of the above eight samples was tested for TPA content, and the blank absorption was subtracted. The data obtained is shown in figure 10.
It can be seen from the concentration of TPA product that the concentration of TPA in PD-MD-hA’s medium is higher than that in PD’s medium or MD’s medium. This strongly supported the engineering success of our three-enzyme complex construction.

Fig.3 standard curve of TPA at OD240

Fig.4 The concentration of TPA product in each experiment group

Scanning electron microscopy

To further confirm the activity of the three-enzyme complex we constructed, we also selected PET plastic sheets in E.coli BL21, E.coli BL21/pET28a-PD, E.coli BL21/pET28a-M, E.coli BL21/pET28a-PD-MD-hA medium to be examined by scanning electron microscopy The results were consistent with expectations. The plastic sheet treated with either E.coli BL21 medium or MD medium has almost no scratches or holes. The plastic sheet after PD treatment has some obvious scratches, while the plastic sheet after PD-MD-hA treatment showed surface covered with scratches, and at the same time, densely packed with holes of various shapes. This scanning electron microscopy further proved that the three-enzyme complex we constructed has a better PET plastic degradation activity than single-enzyme degradation.

Fig.5 Scanning electron microscopy (a) PET plastic sheets treated in E.coli BL21. (b) PET plastic sheets treated in E.coli BL21/pET28a-PD. (c) PET plastic sheets treated in E.coli BL21/pET28a-M. (d) PET plastic sheets treated in E.coli BL21/pET28a-PD-MD-hA medium.

Briefly, we successfully constructed three-enzyme complex as expected, measured the complex's enzyme activity, and performed scanning electron microscopy and TPA detection experiments. The degradation effect of the three-enzyme complex on PET plastic was evaluated qualitatively and quantitatively.
Through the qualitative test of scanning electron microscopy, the enzyme complex created in our project has better effect on PET plastic sheets than PETase alone. The use of PETase alone will only cause scratches on the surface of the plastic sheet. However, our enzyme complex has exceeded PETase activity, causing more significant scratches and on top of that, producing many deep holes. The results of scanning electron microscopy also indirectly accounted for the role of hydrophobin 4 in the enzyme complex. With the presence of hydrophobin 4, the enzyme complex can bind to the surface of the PET plastic sheet. Therefore, instead of dispersing in water and induce reaction through random collisions, the enzyme is more likely to dig deeper into the surface.
Through the quantitative test of TPA production detection, we found that the degradation effect of our enzyme complex is better than PETase used alone, and the degradation efficiency has been increased by two times.