Upcycling soybean pulp for sustainable amino acid and subsequent protein biomanufacturing via a one-pot thermophilic protease cascade treatment

Amino acids are essential components of culture media for fermentative protein production as de novo amino acid synthesis is highly energy-consuming for the host. Soybean pulp (i.e., okara) is a low-value byproduct from soybean processing; however, okara is rich in insoluble proteins. Therefore, okara could be a sustainable source of amino acids for fermentative protein production. Conventional industrial methods for amino acid harvesting employ high-temperature acidic proteolysis to hydrolyze protein sources. However, these conditions are harsh and the process is time-consuming. To increase throughput and yield, we developed a one-pot, two-protease cascade capable of complete okara proteolysis into oligopeptides and individual amino acids in 3 h. Interestingly, we observed an unprecedented synergy between the thermophilic endopeptidase (alcalase) and hyperthermophilic exopeptidase (TETamp), which allows the two-protease cascade to function optimally at 60 °C and pH 7.5. Unlike the conventional method, the enzymatic process preserves tryptophan and asparagine, resulting in an almost complete recovery of total amino acid equivalent from okara. Furthermore, both E. coli and Bacillus megaterium cultures cultivated in the enzymatic okara hydrolysates demonstrated comparable GFP yields compared to those cultivated in LB medium, respectively. We also used the enzymatic okara hydrolysates for fermentative production of the two proteases used in the enzymatic proteolysis. The cell-lytic activity of alcalase even allows okara proteolysis directly using protease-expressing B. megaterium whole-cell biocatalyst, bypassing the costly protease purification step. In conclusion, this study represents a renovated circular bioeconomy model that converts abundant and low-value agro-waste into sustainable feedstocks of the biotechnological industry.