Difference between revisions of "Part:BBa K4613023"

Latest revision as of 15:32, 12 October 2023

pET-46 Ek_LIC-ADH3

The composite part was constructed to achieve solube expression of ADH3 and analyze the function of ADH3. The composite part can be directly imported into plasmid and express ADH3 induced with IPTG.

We obtained the plasmid pET46_EKLIC-ADH3 from Associate Professor Longhai Dai of Hubei University, ADH3 was expressed by E. coli BL21(DE3) using LB medium. After overnight incubation at 20℃, ADH3 (43.4 kDa) was purified. The purified protein was verified by SDS-PAGE. The purified protein was verified by SDS-PAGE. After that, obvious target bands can be seen at 43.4 kDa and 73.6 kDa shown in Fig. 1c (lanes 4 and 5).

Fig. 1 Results of pET46_EKLIC-ADH3 and pET-29a(+)-T3-ADH3. (a) The plasmid map of pET46EKLIC_ADH3. (b) The plasmid map of pET-29a(+)-T3-ADH3. (c) SDS-PAGE analysis of the purified protein ADH3 in E. coli BL21(DE3) cultured in LB medium express protein for 12 h at 20℃. Lane M: protein marker. Lanes 1-9: flow through and elution containing 10, 20, 20, 50, 50, 100, 100, 250, 250 mM imidazole, respectively. (d) SDS-PAGE analysis of protein expression trials in E. coli BL21(DE3) cultured in LB medium for 12 h using pET-29a(+)-T3-ADH3. Lane M: protein marker. Lanes 1-6: flow through and elution containing 50, 50, 20, 20, 10 mM imidazole, respectively.

To degrade Ochratoxin A (OTA) in a more efficient way, we chose two enzymes, Carboxypeptidase A (CPA) and ADH3. We used the methods described by Xiong L et al. (1992) to assay CPA and ADH3 activity. Fig. 2 shows that the activity of CPA and ADH3. ADH3 was estimated at approximately 1.939 unit. CPA was estimated at approximately 0.646 unit. These results indicated that ADH3 exhibited 3.0-fold higher activity than CPA.

  Fig. 2 Assay of ADH3 and CPA activity. The reaction mixture containing 290 μl of 25 mM Tris buffer, 500 mM NaCl (pH 7.5), 3.26 mg/mL Hippuryl-L-phenylalanine (HLP), and 10 μl of ADH3 dissolved in 20 mM Tris-HCl (pH 8.0), 10 μl of CPA dissolved in 1 M NaCl (pH 8.4) in eppendorf tube was incubated at 25℃ for 5 min.

Moreover, we used High-Performance Liquid Chromatography (HPLC) to determine the detoxification rate of CPA and ADH3 against OTA. The HPLC chromatograms of degradation products of OTA were shown in Fig. 3. The retention times (RT) of OTA and its degradation product was 1.650 min (CPA), 1.652 min (ADH3) and 0.691 min (CPA), 0.709 min (ADH3). After the treatment of OTA with CPA and ADH3, the peak area of OTA decreased significantly compared with the control group, and the new product appeared at 0.692 min (CPA), 0.709 min (ADH3). The detoxification rates of CPA and ADH3 were 98.9% and 100%. It proved that CPA and ADH3 can degrade OTA to OTα. ADH3 gave a better performance in degrading than CPA because it took less reaction time to degrade OTA completely in higher concentrations.

Fig. 3 High performance liquid chromatography (HPLC) chromatogram retention time of OTA and OTα. a.10 μg/mL OTA after incubation with methanol solution(control). b.HPLC chromatogram of degradation products of OTA after incubation with 5 U/mL M-CPA for 24 h. c. 50 μg/mL OTA after incubation with methanol solution(control). d. HPLC chromatogram of degradation products of OTA after incubation with 5 U/mL ADH3 for 30 min.

Reference

1. Dai L, Niu D, Huang J W, et al. Cryo-EM structure and rational engineering of a superefficient ochratoxin A-detoxifying amidohydrolase[J]. Journal of Hazardous Materials, 2023: 131836.

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