胺基酸對於發酵製造蛋白質來說，在微生物培養基中是必不可少的成分，因為對宿主來說從頭合成胺基酸是高度耗能的。豆渣為大豆加工的低價值副產物，但豆渣含有豐富的不可溶性蛋白質，也難以被直接利用；因此，本研究將豆渣作為發酵製造蛋白質的永續胺基酸來源。傳統工業方法通常使用高溫酸性水解來水解蛋白質以獲取胺基酸:然而，其條件嚴苛且過程耗時。為了提高產量和產能，我們開發了一鍋式、兩種蛋白酶串聯的方法，能夠在3小時內完全將豆渣水解為寡肽和胺基酸。在實驗過程中我們觀察到嗜熱內肽酶 (Alcalase)和超嗜熱外肽酶 (TET aminopeptidase)之間的協同作用，使得這兩種蛋白酶串聯在60°C和pH 7.5的條件下能夠達到最佳效果，與傳統方法不同在於可從豆渣中回收大部分的胺基酸，並酵素水解能夠保留色胺酸和天門冬醯胺。並且，培養在豆渣酵素水解物中的E. coli和B. megaterium之綠色螢光蛋白產量與LB培養基中的產量相當。此外，我們還利用豆渣酵素水解物來發酵生產本研究所開發之蛋白酶，也因為Alcalase有著使細胞溶解之活性以至於能夠使用表達蛋白酶之細胞直接在60°C下進行豆渣蛋白水解，避免了昂貴且耗時的蛋白酶純化步驟。綜上所述，本研究提出了一個更新的循環生物經濟模型，將富含蛋白質而低價值的農業廢棄物轉化為永續的生物技術工業原料。;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 hours. Interestingly, we observed an unprecedented synergy between the thermophilic endopeptidase (Alcalase) and hyperthermophilic exopeptidase (TET aminopeptidase), 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 B. 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.
