Part:BBa_K3941001
EGII
Summary
BBa_K3941001 is a codon-optimized (for E.coli DH5âº) 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 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 design of the EGII
262 to 590 and 765 to 1692 nucleotide sequences from Trichoderma reesei were used in the project since the EGII gene is too massive to work on. Signal peptides were removed since enzyme release is unnecessary for the project. Parts such as CBM1 and linkers were removed because of the augmentation of protein folding probability which is a complicated process for a prokaryotic organism. Histidine tag was added at the end of the AA sequence which made protein purification easier.
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 is visible
Lastly we conducted a CMCase Activity Analysis. EGII has an absorbance of 0,595 and enzyme activity of 24,85 U/min.
Figure 5: Calibration Curve for CMCase Activity Analysis
Figure 6: Graphs of EGII'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]
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