Difference between revisions of "Part:BBa K3990015"
(→‎Usage and Biology)
Line 9: Line 9:
 
RoxB (Xanthomonas sp. strain 35Y) is distantly related to RoxAs and resembles RoxAs with respect to molecular mass (70.3 kDa for mature protein) and cofactor content (2 c-type hemes). However, RoxB differs from all currently known RoxAs in having a distinctive product spectrum of C20, C25, C30, and higher oligo-isoprenoids that has been observed only for Lcps so far.
 
RoxB (Xanthomonas sp. strain 35Y) is distantly related to RoxAs and resembles RoxAs with respect to molecular mass (70.3 kDa for mature protein) and cofactor content (2 c-type hemes). However, RoxB differs from all currently known RoxAs in having a distinctive product spectrum of C20, C25, C30, and higher oligo-isoprenoids that has been observed only for Lcps so far.
  
−
Attempts to express RoxB in E.coli were not successful yet[1]. We therefore integrated RoxB under the control of an l-rhamnose-inducible promoter into the genome of a ΔroxA background of Xanthomonas sp. strain 35Y. And the clonies grew on opaque polyisoprene latex overlay (LOV) agar that had been supplemented with 0.1%. After a week, the agar plates were stained with fuchsin reagent, large and intensively stained pink-blue zones appeared around RoxB-expressing colonies(Fig.1A).  
+
Attempts to express RoxB in E.coli were not successful yet. We therefore integrated RoxB under the control of an l-rhamnose-inducible promoter into the genome of a ΔroxA background of Xanthomonas sp. strain 35Y. And the clonies grew on opaque polyisoprene latex overlay (LOV) agar that had been supplemented with 0.1%. After a week, the agar plates were stained with fuchsin reagent, large and intensively stained pink-blue zones appeared around RoxB-expressing colonies(Fig.1A).  
 
It is clear that RoxB has a more effective enzymatic activity than RoxA, when fuchsin solution was added to cell-free culture supernatants of RoxA- or RoxB-overexpressing strains that had been allowed to react with added polyisoprene latex for 2 h(Fig.1B).
 
It is clear that RoxB has a more effective enzymatic activity than RoxA, when fuchsin solution was added to cell-free culture supernatants of RoxA- or RoxB-overexpressing strains that had been allowed to react with added polyisoprene latex for 2 h(Fig.1B).
  
  
 
<img src="https://2021.igem.org/wiki/images/0/06/T--SMS_Shenzhen--RoxB1.png" style="wight:50px;"></img>
 
<img src="https://2021.igem.org/wiki/images/0/06/T--SMS_Shenzhen--RoxB1.png" style="wight:50px;"></img>
−
Fig.1 Rubber oxygenase activities of RoxAand RoxB.
+
Fig.1 Rubber oxygenase activities of RoxAand RoxB.[1]
 
<br>
 
<br>
 
Purified RoxB, Lcp-K30, or RoxA were added to polyisoprene latex in an OXY-4 miniapparatus at 30°C. The concentration of dissolved oxygen was monitored over time (A). The blank corresponds to assay buffer without enzymes. As shown in Figure.2A, the presence of RoxB provoked a strong decrease of the dissolved oxygen concentration at room temperature (23°C), which indicates a high rubber oxygenase activity. <br>
 
Purified RoxB, Lcp-K30, or RoxA were added to polyisoprene latex in an OXY-4 miniapparatus at 30°C. The concentration of dissolved oxygen was monitored over time (A). The blank corresponds to assay buffer without enzymes. As shown in Figure.2A, the presence of RoxB provoked a strong decrease of the dissolved oxygen concentration at room temperature (23°C), which indicates a high rubber oxygenase activity. <br>
Line 22: Line 22:
  
 
<div><img src="https://2021.igem.org/wiki/images/0/04/T--SMS_Shenzhen--RoxB2.png" style="wight:50px;"></img src></div>
 
<div><img src="https://2021.igem.org/wiki/images/0/04/T--SMS_Shenzhen--RoxB2.png" style="wight:50px;"></img src></div>
−
Fig.2 Activity assay of purified rubber oxygenases.
+
Fig.2 Activity assay of purified rubber oxygenases.[1]
 
<br>
 
<br>
  
−
 
+
<h5>Reference</h5>
 +
[1]Birke, J., Röther, W., & Jendrossek, D. (2017). RoxB Is a Novel Type of Rubber Oxygenase That Combines Properties of Rubber Oxygenase RoxA and Latex Clearing Protein (Lcp). Applied and environmental microbiology, 83(14), e00721-17. https://doi.org/10.1128/AEM.00721-17
  
  

Revision as of 20:55, 21 October 2021


RoxB

Rubber cleavage enzyme, the rubber oxygenase RoxBXsp (RoxB of Xanthomonas sp. strain 35Y)

Usage and Biology

RoxB (Xanthomonas sp. strain 35Y) is distantly related to RoxAs and resembles RoxAs with respect to molecular mass (70.3 kDa for mature protein) and cofactor content (2 c-type hemes). However, RoxB differs from all currently known RoxAs in having a distinctive product spectrum of C20, C25, C30, and higher oligo-isoprenoids that has been observed only for Lcps so far.

Attempts to express RoxB in E.coli were not successful yet. We therefore integrated RoxB under the control of an l-rhamnose-inducible promoter into the genome of a ΔroxA background of Xanthomonas sp. strain 35Y. And the clonies grew on opaque polyisoprene latex overlay (LOV) agar that had been supplemented with 0.1%. After a week, the agar plates were stained with fuchsin reagent, large and intensively stained pink-blue zones appeared around RoxB-expressing colonies(Fig.1A). It is clear that RoxB has a more effective enzymatic activity than RoxA, when fuchsin solution was added to cell-free culture supernatants of RoxA- or RoxB-overexpressing strains that had been allowed to react with added polyisoprene latex for 2 h(Fig.1B).


<img src="T--SMS_Shenzhen--RoxB1.png" style="wight:50px;"></img> Fig.1 Rubber oxygenase activities of RoxAand RoxB.[1]
Purified RoxB, Lcp-K30, or RoxA were added to polyisoprene latex in an OXY-4 miniapparatus at 30°C. The concentration of dissolved oxygen was monitored over time (A). The blank corresponds to assay buffer without enzymes. As shown in Figure.2A, the presence of RoxB provoked a strong decrease of the dissolved oxygen concentration at room temperature (23°C), which indicates a high rubber oxygenase activity.
And the products were solvent extracted and analyzed by HPLC (Figure2.B). A mixture of C20, C25, and higher oligo-isoprenoids with terminal aldehyde and keto groups were identified in the oxidized product of RoxB, which is in contrast to RoxA. Therefore, RoxB was proposed to be the first member of a third class of rubber oxygenases (RoxB type) that combines properties of both previously characterized classes of rubber oxygenases (RoxAs and Lcps).


<img src="T--SMS_Shenzhen--RoxB2.png" style="wight:50px;"></img src>

Fig.2 Activity assay of purified rubber oxygenases.[1]

Reference

[1]Birke, J., Röther, W., & Jendrossek, D. (2017). RoxB Is a Novel Type of Rubber Oxygenase That Combines Properties of Rubber Oxygenase RoxA and Latex Clearing Protein (Lcp). Applied and environmental microbiology, 83(14), e00721-17. https://doi.org/10.1128/AEM.00721-17


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1937
    Illegal BamHI site found at 186
    Illegal BamHI site found at 1386
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1078
    Illegal NgoMIV site found at 1429
    Illegal NgoMIV site found at 1465
    Illegal AgeI site found at 598
  • 1000
    COMPATIBLE WITH RFC[1000]