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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Thermostable aminopeptidase from Pyrococcus horikoshii
1National Institute of Bioscience and Human-Technology, Tsukuba, Ibaraki, Japan.
FEBS Letters
|April 28, 1999
Summary
Researchers identified a novel thermostable aminopeptidase from Pyrococcus horikoshii. This enzyme efficiently removes N-terminal amino acids from peptides at high temperatures, aiding in peptide sequence analysis.
Area of Science:
- Biochemistry
- Enzymology
- Extremophile Biology
Background:
- Genomic analysis of thermophilic archaea can reveal novel enzymes with unique properties.
- Pyrococcus horikoshii is a hyperthermophilic archaeon known for its robust enzymes.
Purpose of the Study:
- To identify and characterize a novel protein from Pyrococcus horikoshii with potential enzymatic activity.
- To investigate the biochemical properties, including substrate specificity and optimal conditions, of the expressed protein.
Main Methods:
- Bioinformatic analysis of Pyrococcus horikoshii genome to identify potential enzyme-encoding genes.
- Cloning and expression of the identified gene in Escherichia coli.
- Biochemical assays to determine enzyme activity, stability, and cofactor requirements.
Main Results:
- An open reading frame encoding a 332-amino acid protein with homology to known peptidases was identified.
- The expressed protein exhibited significant aminopeptidase activity, cleaving N-terminal amino acids from various peptide substrates.
- The enzyme demonstrated remarkable thermostability, remaining active at temperatures above 90°C, with optimal activity at this temperature. Cobalt ions were essential for activity, while calcium ions were not.
Conclusions:
- A novel, highly thermostable aminopeptidase was successfully characterized from Pyrococcus horikoshii.
- This enzyme's ability to function at extreme temperatures and cleave N-terminal residues makes it a valuable tool for high-temperature peptide analysis, particularly for removing N(alpha)-acylated residues.
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