Part:BBa_K118022

cex coding sequence encoding Cellulomonas fimi exoglucanase

The cellulolytic bacterium Cellulomonas fimi uses an exoglucanase (from cex, accession M15824) along with 3 endoglucanases in the degradation of cellulose into cellobiose, before use B-glucosidase to catalyse the conversion of cellobiose to D-glucose.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 524
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 157
  Illegal NgoMIV site found at 530
  Illegal NgoMIV site found at 1032
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 577
  Illegal SapI.rc site found at 660
  

Contribution

• Group: [http://2018.igem.org/Team:UESTC-China iGEM Team UESTC-China 2018]
• Author: Liang Zhao, Yetao Zou
• Summary: Enzyme digestion and enzyme activity assay

Characterization from iGEM18-UESTC-China

Molecular weight

This gene codes for a protein of 485 amino acids with a molecular mass of 51.2 kDa.

Enzyme digestion

We did a codon optimization of this part before using it. And we verified it by enzyme digestion.

Fig. 1Fig.1 Double enzyme digestion of BBa_K118022. Lane 1: BBa_K118022 digested by EcoRⅠ+PstⅠ. Lane 2: BBa_K118022 digested by Eco32Ⅰ+PstⅠ.

Filter paper assay

We constructed a plasmid containing cenA gene BBa_K118023, cex gene BBa_K118022. We transformed this plasmid into BL21(DE3). We used an intracellular fraction (crude enzyme solution) obtained by ultrasonication to carry out an experiment for measuring total enzyme activities by the method of filter paper assay.The result is shown on Fig. 2[1].

Fig. 2 Enzyme activity of the total cellulase at pH7.0, 40 ℃.

References

[1]Luciano Silveira MH, Rau M, Pinto da Silva Bon E & Andreaus J. 2012. A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper. Enzyme and Microbial Technology, 51: 280-285.

Contribution

• Group: iGEM Team CAU-China 2019
• Author: Liang siwen, Yan xueshan
• Summary: Enzyme activity assay (CMC-Na as substrate)

Characterization from iGEM19_CAU_China

We linked the cex gene BBa_K118022 into a pET30a(+) backbone, which contains lacZ fragment so that the heterogeneous proteins can be induced by IPTG. The plasmids were transferred into BL21(DE3) strain and we induced the recombinant overnight under the condition of 16℃ 0.08 mM. The expression of the fusion protein was determined by SDS-PAGE (Figure 3).

Fig. 3 SDS-PAGE assay for Cex expression, lane 2

Enzyme Activity Assay

The activity of Cex was detected by measuring the cellulose degradation ability using CMC-Na as the substrate. We employed the procedures used by UESTC-China yet under the condition of citric acid-sodium citrate buffer with pH 4.8 and 50 ℃ for reaction temperature. The cells were disrupted by ultrasonication and the suspension of the centrifugal cell contents was used as the crude enzyme. We measured the cellulose degradation abilities of the crude enzyme.(Figure 4)

Fig. 4 Activity assay for Cex crude enzyme fluid

Compared with the negative control(thermal deactivated crude enzyme fluid), the activity of Cex