Coding

Part:BBa_K1499502

Designed by: Jovita Byemerwa   Group: iGEM14_StanfordBrownSpelman   (2014-09-30)

Cellulose Acetate Esterase (Cae)

Cellulose Acetate Esterase is an enzyme that de-acetylates cellulose acetate by hydrolyzing the acetyl ester moieties in cellulose acetate. The gene sequence is obtained from a bacterium, Neisseria Sicca that uses cellulose acetate as its sole source of carbon. Purified Cellulose Acetate Esterase has a high activity on Cellulose Acetate, but it has been shown to have activity on p-nitrophenyl acetate and some other p-nitrophenyl esters. Since industrially made cellulose acetate is a plastic that tends to take a long time to biodegrade in the environment, the use of this enzyme could help in rapid degradation of cellulose acetate by removing the acetate groups. This part could be used specifically for de-acetylating cellulose acetate and other related esters.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal suffix found in sequence at 1533
    Illegal EcoRI site found at 25
    Illegal XbaI site found at 40
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 25
    Illegal NheI site found at 1384
    Illegal SpeI site found at 1534
    Illegal PstI site found at 1548
    Illegal NotI site found at 31
    Illegal NotI site found at 1541
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 25
    Illegal BamHI site found at 625
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 25
    Illegal suffix found in sequence at 1534
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 25
    Illegal XbaI site found at 40
    Illegal SpeI site found at 1534
    Illegal PstI site found at 1548
    Illegal NgoMIV site found at 588
    Illegal AgeI site found at 326
    Illegal AgeI site found at 499
    Illegal AgeI site found at 730
    Illegal AgeI site found at 912
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

Verification of Part

The part was transformed in E.coli and the protein was purified. A protein gel was run on the purified protein in order to verify that the esterase protein was present. The gel showed a band at 43kDa which is the characteristic molecular mass of the protein.

Figure 1. The Esterase enzyme at 43kDa

Results

Once we had purified the protein, we were able to carry out functional assays on the enzyme. We used cellulose binding dye that selectively binds to cellulose and not cellulose acetate to test whether the enzyme was degrading commercially available cellulose acetate to cellulose. As it can be seen in the image below, over time, the esterase protein was working to degrade cellulose acetate as the intensity of the blue stain was increasing.

Figure 2.

References

1. Sakai K; Yamauchi T; Nakasu F; Ohe T. (1996) Biodegradation of cellulose acetate by Neisseria sicca. Bioscience Biotechnology Biochemistry.(10):1617-22. http://www.ncbi.nlm.nih.gov/pubmed/

2. Sakai K. et al. (1999) Purification and characterization of an esterase involved in cellulose acetate degradation by Neisseria sicca SB. Bioscience Biotechnology Biochemistry. Oct;63(10):1708-13. http://www.ncbi.nlm.nih.gov/pubmed/10586499


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