Part:BBa_K5117002

BpEglA

This parts contains the eglA gene of Bacillus pumilus including its native signal peptide for secretion, encoding an endoglucanase (EC 3.2.1.4).

BpEglA 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 cellulose

Design

For compatibility with the BioBrick RFC[10] standard, the restriction sites EcoRI, XbaI, SpeI, PstI and NotI were removed from the CDS. 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
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 404
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Enzyme characterization according to literature

In the study by Lima et al. (2005), titled "Molecular characterization of a β-1,4-endoglucanase from an endophytic Bacillus pumilus strain", the researchers cloned the eglA gene from a citrus endophytic Bacillus pumilus strain. This gene encodes a β-1,4-endoglucanase capable of hydrolyzing cellulose under in vitro conditions. The enzyme, expressed as a His-tag fusion protein in E. coli, was purified using immobilized metal affinity chromatography (IMAC). SDS-PAGE analysis estimated the molecular weight of the purified protein to be approximately 71.3 kDa (Lima et al. 2005).

The enzyme demonstrates optimal activity within a neutral pH range of 5 to 8 when using carboxymethylcellulose (CMC) as a substrate. It shows no activity at pH 4 and about 30 % activity at pH 9. The enzyme retains full activity after a 3-hour incubation at pH 6.2 to 8.6 and maintains 53 % activity at pH 9.6. Purified EglA remains active even after extended incubation periods of 6, 12, and 24 hours in the pH range of 6 to 8 (Lima et al. 2005).

The enzyme exhibits maximum activity at 60 °C and shows exceptional thermostability. It retains more than 90% of its activity after 6 hours at 60 °C and after 24 hours at 50 °C. This heat stability, combined with its broad pH tolerance, positions EglA as a promising candidate for biotechnological applications, particularly in processes that require stable enzyme activity at temperatures of 50 to 60 °C over long periods. The enzyme's ability to maintain significant activity under these conditions highlights its potential utility in industrial processes involving cellulose degradation (Lima et al. 2005).

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

• Gene sequence: https://www.ncbi.nlm.nih.gov/nuccore/AY339624
• Protein sequence: https://www.ncbi.nlm.nih.gov/protein/AAQ91573
• UniProtKB: https://www.uniprot.org/uniprotkb/Q5YLG1/entry

References

Lima A. O., Quecine M. C., Fungaro M. H., Andreote F. D., Maccheroni W., Araújo W. L., Silva-Filho M. C., Pizzirani-Kleiner A. A., Azevedo J. L. (2005): Molecular characterization of a β-1, 4-endoglucanase from an endophytic Bacillus pumilus strain. Applied microbiology and biotechnology 68, 57-65. https://doi.org/10.1007/s00253-004-1740-1