Part:BBa_K3941002
EGII (C99V)
BBa_K3941002 is a codon-optimized (for E. coli DH5âº) C99V mutated version of the endoglucanase (EG) gene that cleaves the internal beta-1,4-glycosidic bonds in cellulose. We optimized the sequence for expression and added a 6XHis at the end.
Figure 1: Codon optimized EGII with a C99V mutation and a His-Tag
Introduction
EGII is produced by Trichoderma reesei which is a fungi. Substrate specificity, binding properties, and cleavage products of EGII were examined to evaluate its potential multiple enzymatic activities.
Design
Figure 2: Schema of how did we designed our EGII (C99V)
We mutated the EGII to use and later on optimized it to use in E. coli DH5⺠bacteria. We also used E. coli BL21 to express our plasmids. To harvest our proteins, we added a His-tag (6XHis) at the end of our AA sequence. We changed the Cystine from the 99th amino acid to Valine. This mutation decreases the number of disulfide bonds. Decreasing the number of disulfide bonds increases the flexibility of protein and higher thermal stability.
Results
We have done a spectrophotometer absorbance analysis.
Figure 3: The results of spectrophotometer absorbance analysis. The numerical columns are A230, A260, A280, A320, A260/A280, A260/A230 respectively
After that we have done an agarose gel electrophoresis.
Figure 4: The comparision between the backbone of the plasmid and EGII (C99V) is visible
Lastly we conducted a CMCase Activity Analysis. EGII(C99V) has an absorbance of 0,051 and enzyme activity of 2e-2 umol/L.min.
Figure 5: Calibration Curve for CMCase Activity Analysis
Figure 6: Graphs of EGII (C99V)'s CMCase Activity Analysis
References
- Tjandra, Kezia & Sari Dewi, Kartika & Fuad, Asrul Muhamad & Anindyawati, Trisanti. (2020). Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter. Indonesian Journal of Biotechnology. 25. 127. 10.22146/ijbiotech.55604.
- Akbarzadeh, Ali & Pourzardosht, Navid & Dehnavi, Ehsan & Siadat, Seyed & Zamani, Mohammadreza & Motallebi, Mostafa & Jamnani, Farnaz & Aghaeepoor, Mojtaba & Barshan-tashnizi, Mohammad. (2018). Disulfide bonds elimination of endoglucanase II from Trichoderma reesei by site-directed mutagenesis to improve enzyme activity and thermal stability: An experimental and theoretical approach. International Journal of Biological Macromolecules. 120. 10.1016/j.ijbiomac.2018.09.164.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |