Part:BBa_K5117007
BhBglA
This part contains the bglA gene of Bacillus halodurans, encoding a β-glucosidase (EC 3.2.1.21).
BhBglA only served for design purposes of the TU Dresden iGEM 2024 Team and was required for the construction of composite parts (see Contribution page).
Biosafety level: S1
Target organism: Bacillus subtilis
Main purpose of use: Expression in the host B. subtilis
Potential application: Degradation of cellobiose
Design
For compatibility with the BioBrick RFC[10] standard, the restriction sites EcoRI, XbaI, SpeI, PstI and NotI were removed from the coding sequence. To make the part compatible with the Type IIS standard, BsaI and SapI sites were removed as well. This was achieved by codon exchange using the codon usage table of Bacillus subtilis (Codon Usage Database Kazusa).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 564
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Enzyme characterization according to literature
In the study by Naz et al. (2010) titled "Enhanced production and characterization of a beta-glucosidase from Bacillus halodurans expressed in Escherichia coli", the production and characterization of the enzyme β-glucosidase A (BglA) enzyme from Bacillus halodurans was explored through heterologous expression in E. coli (Naz et al. 2010).
The recombinant plasmid pET-BglA was transformed into E. coli BL21(DE3) CodonPlus cells. The induced protein was detected by SDS-PAGE, where a prominent band corresponding to the expected molecular weight of ≈ 51 kDa was observed (Naz et al. 2010).
Cells were harvested, sonicated, and the soluble fraction was precipitated using ammonium sulfate. Purification was performed using anion exchange chromatography. The fractions were analyzed for BglA activity, yielding a purified enzyme for further characterization. BglA displayed the highest activity at pH 8.0, using glycine-NaOH buffer. BglA was stable at pH 7.5 – 8.0 at 45 °C for one hour, but stability decreased significantly at higher pH levels (Naz et al. 2010).
The enzyme retained more than 90% residual activity when incubated at pH 7.5-8.0 at 45 °C for one hour, but activity sharply decreased at pH > 8.0, dropping to 20% residual activity at pH of 9.5. The optimal temperature for BglA activity was found to be 45 °C when assayed using p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate. The enzyme retained 80 % of its activity when incubated at temperatures up to 45 °C for 1 hour, but activity decreased significantly at higher temperatures. BglA retained 80 % activity when incubated at 45 °C for one hour, but a decline in activity was observed with longer incubation times or at higher temperatures (Naz et al. 2010).
More information related to this part can be found in the following publications and databases:
- Takami, H., Nakasone, K., Takaki, Y., Maeno, G., Sasaki, R., Masui N., Fuji F., Hirama C., Nakamura Y., Ogasawara N., Kuhara S., Horikoshi, K. (2000): Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucleic acids research 28(21), 4317-4331. https://doi.org/10.1093/nar/28.21.4317
- Gene sequence: https://www.ncbi.nlm.nih.gov/nuccore/BA000004
- Protein sequence: https://www.ncbi.nlm.nih.gov/protein/BAB05642
- UniProtKB: https://www.uniprot.org/uniprotkb/Q9KBK3/entry
References
Naz S., Ikram N., Rajoka M. I., Sadaf S., Akhtar M. W. (2010): Enhanced production and characterization of a β-glucosidase from Bacillus halodurans expressed in Escherichia coli. Biochemistry (Moscow) 75, 513-518. https://doi.org/10.1134/s0006297910040164
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