The key proteolytic enzyme analysis of industrial Aspergillus oryzae at solid-state koji fermentation with a local database extension

The productivity and flavor quality of soy sauce are partly determined by the proteolytic enzymes secreted by the industrial microbe Aspergillus oryzae, which break down proteins into peptides and amino acids. Because of how complicated it is to classify proteolytic enzymes and understand how they work, not much is known about what is in the profiles of proteolytic enzymes and which enzymes are most important to A. oryzae during solid-state fermentation. In this study, we collected industrial A. oryzae from Chinese and Japanese soy sauce production, both of which have excellent protein hydrolysis capability but have numerous genetic differences. We systematically analyzed the in vitro enzyme profiles of two industrial A. oryzae based on 4D label-free proteome sequencing technology. In addition, we constructed a local database of proteolytic enzymes from the Aspergillus genus, which helps us analyze the proteomics data more precisely. We found that 96 proteolytic enzymes are excreted by industrial A. oryzae, of which 27 are endopeptidases and 39 are exopeptidases. Based on the relative protein expression and transcription quantity, as well as the catalytic characteristics, we identified 27 potential key proteolytic enzymes associated with high protein breakdown efficiency at solid-state fermentation. Meanwhile, we proposed eight proteolytic enzymes with potential hydrolytic activity. This study deepens our understanding of the proteolytic enzyme profiles excreted by A. oryzae at solid state and provides our knowledge of the candidate key proteolytic enzymes during koji fermentation.