Prediction, biochemical characterization and application of key proteolytic enzymes from aspergillus oryzae BL18 in soy sauce fermentation

Abstract

Aspergillus oryzae is the most widely used starter in koji-making of bean-based fermented foods due to its strong ability to produce proteolytic enzymes. However, the information of the key proteolytic enzymes in A. oryzae is still lacking. This study aimed to mine the key proteolytic enzymes from A. oryzae BL18 through genomic and transcriptomics analysis, to characterize their biochemical properties and to evaluate their application effect in soy sauce fermentation. Thirty-eight proteolytic enzyme genes with secretory ability were predicted while GME1689_g, GME238_g, GME3470_g and GME6033_g genes were found to have high expression levels among these genes during the koji-making process. After that, these four genes were heterologously expressed in Komagataella phaffii GS115 and their biochemical properties were characterized. The results showed that GME1689_g and GME238_g were optimal in neutral condition while GME3470_g and GME6033_g were acidic enzymes. Among these four enzymes, GME1689_g and GME238_g were more salt tolerant and had higher degradation degree towards globulin and albumin. Therefore, these two enzymes were applied in the initial stage of soy sauce fermentation individually or together. Compared to control group, the concentration of free amino acids in soy sauce fermented with addition of proteolytic enzymes were 20-25 % higher while more volatile flavor compounds (such as linolool and octen-3-ol) were enriched in both quantity and concentration. Through sensory evaluation, the addition of proteolytic enzymes, especially GME1689_g, could enhance the flavor of soy sauce with the average sensory score increasing from 7.5 to 8.0-8.5. The results obtained in this study could not only enhance our understanding of the proteolytic enzymes in A. oryzae but also provide possible solution for quality enhancement of soy sauce.

Keywords: Aspergillus oryzae; Enzymatic properties; Heterologous expression; Proteolytic enzymes; Soy sauce.