Difference between revisions of "Part:BBa K4613002"

Latest revision as of 17:22, 11 October 2023

ADH3

ADH3 is an amidohydrolase derived from Stenotrophomonas acidaminiphila and forms an octamer in solution. ADH3 was reported to exhibit 57- to 35,000-fold higher activity than other enzymes and is the most efficient OTA-detoxifying enzyme reported thus far and can hydrolyze OTA to nontoxic ochratoxin α (OTα) and L-β-phenylalanine (Phe). Moreover, soluble protein expression of ADH3 in Escherichia coli has been realized.

Fig. 1 SDS-PAGE analysis of the purified protein ADH3 in E. coli BL21 (DE3) cultured in LB medium express protein for 12 hours 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.

Fig. 2 Assay of ADH3 activity. A 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) in eppendorf tube was incubated at 25℃ for 5 min.

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.

Because of its high efficiency and soluble expression in Escherichia coli , we used the variant S88E of ADH3 engineered by Xiong L et al.(2023) in our project to degrade OTA.

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.

Sequence and Features