A novel non-thermostable deuterolysin from Aspergillus oryzae

Area of Science:

• Microbiology
• Enzymology
• Molecular Biology

Background:

• Aspergillus oryzae possesses multiple putative deuterolysin genes.
• Deuterolysins are metalloproteinases involved in protein degradation.
• Previous research identified the NpII gene, now recognized as deuA.

Purpose of the Study:

• To identify and characterize deuterolysin genes in Aspergillus oryzae.
• To compare the enzymatic properties and substrate specificities of DeuA and DeuB.
• To investigate the transcriptional regulation of deuterolysin genes.

Main Methods:

• Bioinformatic analysis of the Aspergillus oryzae genome.
• Overexpression and purification of recombinant DeuA and DeuB enzymes.
• Enzyme activity assays using protein and peptidyl MCA substrates.
• Thermostability testing of purified enzymes.
• Quantitative reverse transcription PCR for gene expression analysis.

Main Results:

• Three deuterolysin genes (deuA, deuB, deuC) were identified in A. oryzae.
• Recombinant DeuA and DeuB were successfully expressed and purified.
• DeuB demonstrated broader substrate specificity than DeuA.
• DeuB was inactivated above 80°C, unlike the thermostable DeuA.
• Only deuB was expressed in liquid culture, with its transcription upregulated by protein substrates.
• NaNO3 addition mitigated the effect of protein substrates on deuB transcription.

Conclusions:

• Aspergillus oryzae encodes at least two functional deuterolysins, DeuA and DeuB, with distinct enzymatic characteristics.
• DeuB plays a significant role in protein degradation, with its expression tightly regulated by substrate availability.
• Understanding these enzymes provides insights into fungal protease functions and regulation.