Coding

Part:BBa_K5117009

Designed by: Jenny Sauermann   Group: iGEM24_TU-Dresden   (2024-08-31)


AtBglA

This part contains the bglA gene of Acetivibrio thermocellus (synonym Clostridium thermocellum), encoding a β-glucosidase (EC 3.2.1.21).

AtBglA only served for design purposes of the TU Dresden iGEM 2024 Team and was required for the construction of composite parts (see Contribution page).


Target organism: Bacillus subtilis

Main purpose of use: Gene expression and protein production using the host Bacillus subtilis


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Enzyme characterization according to literature

Clostridium thermocellum produces a thermostable enzyme called β-glucosidase A (BglA), which plays a crucial role in cellulose degradation. Two significant studies have characterized this enzyme to assess its potential for industrial applications.

Gräbnitz et al. (1991) conducted a study titled "Structure of the beta-glucosidase gene bglA of Clostridium thermocellum. Sequence analysis reveals a superfamily of cellulases and beta-glycosidases including human lactase/phlorizin hydrolase". The researchers determined the nucleotide sequence of the bglA gene, which encodes a polypeptide of approximately 51 kDa. The enzyme was expressed in E. coli JM83 and purified using heat treatment and ion-exchange chromatography, leveraging its thermostability to separate it from host proteins (Gräbnitz et al. 1991).

The purified β-glucosidase A displayed activity toward p-nitrophenyl β-D-glucopyranoside (pNPG), with enzyme assays measuring the release of p-nitrophenol. The enzyme showed optimal activity at 60°C in citrate/phosphate buffer at pH 6.0 (Gräbnitz et al. 1991).

Ahmed et al. (2019) further explored this enzyme in their study titled "Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable β-Glucosidase from Clostridium thermocellum in Escherichia coli". They aimed to achieve high-level soluble expression of bglA in E. coli. The enzyme was expressed as a partially soluble protein of approximately 53 kDa, with about 50% in the soluble fraction. Purification involved heat inactivation and nickel-affinity chromatography (98 % purity) (Ahmed et al. 2019).

The optimum temperature for the BglA reaction was determined by incubating separate reactions at various temperatures ranging from 30 °C to 75 °C for 30 minutes in 1.5 M Tris-Cl buffer (pH 7). Similarly, the optimum pH for the BglA reaction was identified by conducting reactions across a pH range of 4 to 10. For pH values between 4 and 8, 0.15 M citrate-phosphate buffer was used, while for pH values between 8 and 10, 1.5 M Tris-Cl buffer was used (Ahmed et al. 2019).

To assess temperature stability, the enzyme was incubated for one hour at temperatures between 25°C and 75°C, with intervals of 5°C. pH stability was evaluated by incubating the enzyme at temperatures for 30 minutes in the respective buffers, 0.15 M citrate-phosphate for pH 4 to 8 and 1.5 M Tris-Cl for pH 8 to 10. The remaining enzyme activity (expressed as U/mg) after these treatments provided the temperature and pH stability thresholds for BglA. The enzyme exhibited maximum activity at 50 °C, retaining catalytic function up to 60 °C. BglA displayed optimal activity at pH 7.0, maintaining stability at this pH (Ahmed et al. 2019)..


More information related to this part can be found in the following publications and databases:


References

Ahmed S. S., Akhter M., Sajjad M., Gul R., Khurshid S. (2019): Soluble Production, Characterization, and Structural Aesthetics of an Industrially Important Thermostable β‐Glucosidase from Clostridium thermocellum in Escherichia coli. BioMed Research International 2019(1), 9308593. https://doi.org/10.1155/2019/9308593

Gräbnitz F., Seiss M., Rücknagel K. P., Staudenbauer W. L. (1991): Structure of the β‐glucosidase gene bglA of Clostridium thermocellum: Sequence analysis reveals a superfamily of cellulases and β‐glycosidases including human lactase/phlorizin hydrolase. European journal of biochemistry 200(2), 301-309. https://doi.org/10.1111/j.1432-1033.1991.tb16186.x  


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