Difference between revisions of "Part:BBa K3941001"

Latest revision as of 16:05, 21 October 2021

EGII

Summary

BBa_K3941001 is a codon-optimized (for E.coli) 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

The part’s sequence originated from NCBI GenBank M19373. Substrate specificity, binding properties, and cleavage products of EGII were examined to evaluate its potential multiple enzymatic activities. EGII has a molecular weight of 52 kDa and has an optimum pH of 5.0 and optimum temperature of 40°C and 50°C. EGII can maintain 89% of its endoglucanase activity at 40 °C and more than 80% at 50 °C for 60 min.

Design

Figure 2: Schema of design of the EGII

EGII enzyme is produced in a eukaryotic organism (T. reesei) by default but this part was expressed in a prokaryotic bacterium. It is a difficult process because a prokaryotic bacteria can’t handle too much protein folding. Nucleotide sequences 262-590 and 765-1692 were used because the remaining sequences contain intron regions. 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

Genes from IDT in the cloning plasmid were transformed into strain E.coli and plasmid isolation was performed. 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

To obtain pET29b+EGII plasmid structure, both cloning vector and expression vector digested with the same restriction enzymes. Agarose gel recovery was performed to isolate required DNA bands, inserts and the backbone, from the gel. They ligated and transformed to E. coli BL21 for expression of the protein. Protein expression induction was performed by adding IPTG.

Figure 4: The comparision between the backbone of the plasmid and EGII is visible

Protein concentrations were determined with Bradford after protein isolation.

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 (x: Glucose Concentration (mg/mL), y: Absorbance)

Figure 6: Graphs of EGII's CMCase Activity Analysis (x: Dilution Factor, y: Product (mg))

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