Difference between revisions of "Part:BBa K4613002"
 
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<partinfo>BBa_K4613002 short</partinfo>
 
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ADH3 is an amidohydrolase derived from Stenotrophomonas acidaminiphila and forms an octamer in solution.
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ADH3 is an amidohydrolase derived from <em>Stenotrophomonas acidaminiphila</em> and forms an octamer in solution.
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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 &#945; (OT&#945;) and L-&#946;-phenylalanine (Phe). Moreover, soluble protein expression of ADH3 in Escherichia coli has been realized.
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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 &#945; (OT&#945;) and L-&#946;-phenylalanine (Phe). Moreover, soluble protein expression of ADH3 in <em>Escherichia coli</em> has been realized.
  
  
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<center><img src="https://static.igem.wiki/teams/4613/wiki/parts/parts/parts/pet46-adh3-imidazole-eluzed.jpg"with="1000" height="" width="500" height=""/></center>
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<p style="text-align: center!important;"><b>Fig.1 SDS-PAGE analysis of purified enzymes. M, standard protein markers; 1, purified E. coli–expressed ADH3.</b></p>
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<p style="text-align: center!important;"><b>Fig. 1 SDS-PAGE analysis of the purified protein ADH3 in <i>E. coli</i> 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.</b></p>
  
  
 
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<center><img src="https://static.igem.wiki/teams/4613/wiki/parts/parts/hplc-analysis-of-the-ota-adh3.png"with="1000" height="" width="500" height=""/></center>
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<p style="text-align: center!important;"><b>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.</b></p>
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<p style="text-align: center!important;"><b>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.
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<p style="text-align: center!important;"><b>Fig.2 HPLC analysis of the reaction mixtures catalyzed by WT and variant S88.</b></p>
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Because of its high efficiency and soluble expression in <em> Escherichia coli </em>, we used the variant S88E of ADH3 engineered by <em> Xiong L et al.</em>(2023) in our project to degrade OTA.
  
  
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Because of its high efficiency and soluble expression in <em> Escherichia coli </em>, we used the variant S88E of ADH3 engineered by <em> Xiong L et al. </em>(2023) in our project to degrade OTA.
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==== Reference ====
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#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.
  
  

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 733
    Illegal AgeI site found at 421
    Illegal AgeI site found at 583
  • 1000
    COMPATIBLE WITH RFC[1000]