Kumamolisin from Alicyclobacillus sendaiensis strain NTAP-1 is a serine protease with collagenase activity. After molecular engineering, a kumamolisin mutant, named Kuma030, was obtained with high proteolytic activity against gluten, which might cause celiac disease. Kuma030 exhibited its potential application in industrial and medicine, while challenges remained of its large-scale purification and production. In the studies here, we successfully overexpressed the Kuma030 in E. coli BL21 (DE3) by anchoring a SUMO (Small Ubiquitin-like Modifier) fusion protein at its N-terminal end. In addition, a fast protein purification procedure was developed according to the acidophilic and thermophilic properties of Alicyclobacillus sendaiensis. After a simple acid treatment followed by a heat treatment, a total of 9.9 mg functional Kuma030 was quickly obtained form 1 L LB media culture. This purified Kuma030 was confirmed to be functional to cleave the PQ sequences in a designed protein substrate, and the gluten in actual food samples, such as whole wheat bread and beer, in a fast manner. Our studies provided an efficient strategy for the overexpression and purification of functional Kuma030 in E. coli, which might expand its broad practical applications.

中文翻译：

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通过热酸处理无需色谱法在大肠杆菌中高效生产麸质水解酶 Kuma030

来自仙台脂环杆菌 NTAP-1 菌株的 Kumamolisin 是一种具有胶原酶活性的丝氨酸蛋白酶。经过分子工程，获得了一种名为 Kuma030 的 kumamolisin 突变体，它对可能导致乳糜泻的麸质具有高蛋白水解活性。隈研吾030展现了其在工业和医药方面的潜在应用，但其大规模纯化和生产仍面临挑战。在这里的研究中，我们通过在其 N 末端锚定 SUMO（小泛素样修饰符）融合蛋白，在大肠杆菌 BL21 (DE3) 中成功过表达 Kuma030。此外，根据仙台脂环杆菌的嗜酸和嗜热特性，开发了一种快速蛋白质纯化方法。经过简单的酸处理和热处理，共 9 次。从 1 L LB 培养基培养物中快速获得 9 mg 功能性 Kuma030。经证实，这种纯化的 Kuma030 具有快速切割设计蛋白质底物中 PQ 序列和实际食品样品（如全麦面包和啤酒）中的麸质的功能。我们的研究为大肠杆菌中功能性 Kuma030 的过表达和纯化提供了一种有效的策略，这可能会扩大其广泛的实际应用。