Active subtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence

Area of Science:

• Biochemistry
• Enzymology
• Extremophile Biology

Background:

• Subtilisin-like proteases are widely distributed enzymes.
• Hyperthermophilic archaea possess unique enzymes adapted to extreme conditions.
• Understanding Thermococcus kodakaraensis subtilisin provides insights into thermostable enzyme evolution.

Purpose of the Study:

• To clone and characterize the gene encoding subtilisin-like protease from Thermococcus kodakaraensis KOD1.
• To investigate the biochemical and enzymatic properties of T. kodakaraensis subtilisin.
• To compare the stability and characteristics of T. kodakaraensis subtilisin with bacterial subtilisins.

Main Methods:

• Gene cloning from Thermococcus kodakaraensis KOD1.
• Recombinant protein expression in Escherichia coli.
• Protein purification using SDS-polyacrylamide gel electrophoresis.
• Biochemical assays including substrate hydrolysis and thermal stability tests.

Main Results:

• T. kodakaraensis subtilisin was successfully cloned and expressed.
• The recombinant enzyme, composed of 398 amino acids, functions as a monomer.
• Optimal activity was observed at pH 9.5 and 80°C, requiring Ca(2+).
• The protease demonstrated exceptional thermal stability, with significant half-lives at elevated temperatures.

Conclusions:

• T. kodakaraensis subtilisin is a highly thermostable protease with unique refolding properties compared to bacterial counterparts.
• Its stability and enzymatic characteristics make it a promising candidate for industrial applications requiring high temperatures.
• This research expands the understanding of protease diversity in hyperthermophilic archaea.