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Heterologous expression and characterisation of a biosynthetic thiolase from Clostridium butyricum DSM 10702

Paper ID Volume ID Publish Year Pages File Format Full-Text
17805 42697 2009 6 PDF Available
Title
Heterologous expression and characterisation of a biosynthetic thiolase from Clostridium butyricum DSM 10702
Abstract

Biosynthetic thiolases (EC 2.3.1.9) are key enzymes in the branched catabolism of diverse clostridia as their activity and regulation influence the production of organic acids and solvents. In Clostridium butyricum, they are also involved in the production of hydrogen as a sustainable and environmentally benign energy source. In this study, the gene coding for thiolase from C. butyricum DSM 10702 was cloned by genome walking. It was found to consist of 1179 bp coding for a protein with 393 amino acids and a deduced molecular weight of 41.4 kDa. The enzyme was fused to an N-terminal his-tag, expressed in Escherichia coli, purified to near homogeneity and characterised for biochemical and kinetic properties. Gel filtration chromatography revealed that the catalytically active enzyme consists of a homotetramer. The enzyme showed a KM of ∼32 μM towards acetoacetyl-CoA and of ∼21 μM towards CoASH at 30 °C and pH 8.0. Claisen condensation of acetyl-CoA by thiolase was analysed in a coupled enzyme assay, where β-hydroxybutyryl-CoA dehydrogenase was applied catalysing the subsequent NADH-dependant reduction of the formed condensation product acetoacetyl-CoA. For this purpose the latter enzyme was cloned from C. butyricum DSM 10702 and recombinantly expressed in E. coli. The KM of thiolase towards acetyl-CoA was ∼674 μM at 30 °C and pH 7.5. Acetyl-CoA condensation was inhibited even at micromolar concentrations of CoASH indicating that CoASH has an important regulatory function in vivo.

Keywords
Biosynthetic thiolase; Clostridium butyricum; Claisen condensation; Acetyl-CoA; Acetoacetyl-CoA
First Page Preview
Heterologous expression and characterisation of a biosynthetic thiolase from Clostridium butyricum DSM 10702
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 45, Issue 5, 5 November 2009, Pages 361–366
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering