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Cloning, sequence and expression in Escherichia coli of the gene encoding phosphofructokinase from Bacillus
G J Rouwendal1, L H Zwiers, E J Wolbert
1Department of Bioconversion, Agrotechnological Research Institute (ATO-DLO), Wageningen, The Netherlands. g.j.a.rouwendal@ato.dlo.nl
Abstract:
A chromosomal DNA fragment containing the Bacillus macquariensis (Bm) ATP-dependent phosphofructokinase-encoding gene (pfk) was cloned from a subgenomic library in pUC19 using a PCR-derived probe. The region containing pfk, including flanking sequences, was sequenced and the deduced amino acid sequence (aa) was found to be homologous to other PFK, but it contained two single-aa changes conserved in a range of other organisms from pro- and eukaryotic origins. Enzymatic studies with PFK purified from overproducing Escherichia coli (Ec) host cells showed that the Bm enzyme is similar to B. stearothermophilus (Bs) PFK in many respects and that it is relatively cold stable.
Insights
Researchers cloned and characterized the Bacillus macquariensis (Bm) phosphofructokinase (pfk) gene. The Bm enzyme is cold-stable and shares similarities with other phosphofructokinase enzymes.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Phosphofructokinase (PFK) is a key enzyme in glycolysis.
- Understanding PFK from diverse bacterial species provides insights into enzyme evolution and function.
- Bacillus macquariensis (Bm) PFK has not been extensively characterized.
Purpose of the Study:
- To clone and characterize the ATP-dependent phosphofructokinase (pfk) gene from Bacillus macquariensis.
- To analyze the deduced amino acid sequence and compare it with other PFK enzymes.
- To investigate the enzymatic properties and stability of the purified Bm PFK.
Main Methods:
- Cloning of the Bm pfk gene using a PCR-derived probe and a subgenomic library in pUC19.
- DNA sequencing of the pfk gene and flanking regions.
- Expression and purification of Bm PFK in Escherichia coli (Ec) host cells.
- Enzymatic assays and stability studies of the purified enzyme.
Main Results:
- The Bm pfk gene was successfully cloned and sequenced.
- The deduced amino acid sequence showed homology to other PFKs, with two conserved single-amino acid changes.
- Purified Bm PFK exhibited enzymatic properties similar to Bacillus stearothermophilus (Bs) PFK.
- The Bm PFK enzyme demonstrated relative cold stability.
Conclusions:
- The characterization of Bm PFK provides new information on the diversity of phosphofructokinases.
- The conserved amino acid changes may play a role in enzyme function or stability.
- The cold stability of Bm PFK suggests potential applications in low-temperature biochemical processes.