Part:BBa_K4613002
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°C . Lane M: protein marker. Lanes 1-9: flow through and elution containing 10, 20, 20, 50, 50, 100, 100, 250, 250mm imidazole, respectively.
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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 733
Illegal AgeI site found at 421
Illegal AgeI site found at 583 - 1000COMPATIBLE WITH RFC[1000]
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