L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Enzymes are crucial biocatalysts in industrial processes.
• The demand for stereoselective enzymes, particularly amidases, is growing in fine chemical synthesis.
• Ochrobactrum anthropi harbors potential biocatalysts for industrial applications.

Purpose of the Study:

• To purify and characterize a novel L-specific amidase from Ochrobactrum anthropi.
• To investigate the enzyme's stability, substrate specificity, and cofactor requirements.
• To clone and express the gene encoding the L-amidase for heterologous production.

Main Methods:

• Purification of L-amidase to homogeneity using standard biochemical techniques.
• Enzyme activity assays across a range of pH, temperatures, and substrate concentrations.
• Inhibition studies using protease inhibitors and metal chelators.
• Gene cloning, sequencing, and heterologous expression in Escherichia coli.

Main Results:

• The purified L-amidase is optimally active between pH 6-8.5 and stable up to 60°C.
• The enzyme requires divalent metal ions, with Mn2+ and Mg2+ significantly enhancing activity.
• It exhibits high enantioselectivity (E > 150) for L-amino acid amides and related substrates.
• The gene encodes a 314-amino acid protein, suggesting a homodimeric structure for the native enzyme.
• Efficient heterologous expression was achieved in E. coli.

Conclusions:

• Ochrobactrum anthropi L-amidase possesses unique properties, including high stability and broad substrate scope.
• Its L-enantioselectivity makes it a valuable biocatalyst for chiral synthesis.
• The enzyme's characteristics are highly attractive for applications in the fine-chemicals industry.