Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis

Area of Science:

• Biochemistry
• Enzymology
• Microbiology

Background:

• High-alkaline proteases are crucial enzymes with applications in various industries.
• Understanding the diversity of proteases from extremophilic microorganisms is important for discovering novel biocatalysts.

Purpose of the Study:

• To purify and characterize a novel high-alkaline protease from the strictly anaerobic and extremely alkaliphilic bacterium Alkaliphilus transvaalensis.
• To elucidate the enzymatic properties, substrate specificity, and phylogenetic position of the newly discovered protease.

Main Methods:

• Purification of the high-alkaline protease (ALTP) to homogeneity.
• Enzyme activity assays at various pH and temperature conditions.
• Mass spectrometric analysis of hydrolyzed insulin B-chain to determine cleavage sites.
• Amino acid sequencing and phylogenetic analysis of the subtilase family.

Main Results:

• A novel high-alkaline protease (ALTP) with a molecular mass of 30 kDa was purified.
• ALTP demonstrated maximal caseinolytic activity above pH 12.6 at 40°C.
• The enzyme exhibited widespread proteolytic functions, hydrolyzing 24 out of 29 peptide bonds in oxidized insulin B-chain.
• Phylogenetic analysis placed ALTP in a new subcluster within the subtilase family A, distinct from known subtilisins and high-alkaline proteases.
• ALTP is the first high-alkaline protease identified from a strict anaerobe in this family.

Conclusions:

• Alkaliphilus transvaalensis harbors a unique high-alkaline protease (ALTP) with broad substrate specificity.
• ALTP represents a novel enzyme class, expanding the known diversity of proteases from anaerobic extremophiles.
• This discovery opens avenues for exploring new biotechnological applications of proteases under extreme alkaline conditions.