Protein glutaminase (PG) is an enzyme that specifically catalyzes the deamidation of glutamine residues on proteins or peptides, remarkably improving the solubility, emulsification and foaming properties of food proteins and, thereby, conferring great potential in food industry applications. PG is primarily produced from wild strains of Chryseobacterium proteolyticum and the low enzyme production yield restricts large‐scale industrial applications. In this context, by evaluating different cleavage site insertions between the pro‐region and mature domain of PG as well as different linkers flanking the cleavage site, an E. coli expression and purification protocol has been developed to produce active recombinant PG. To simplify the production workflow, we developed a sequential dual expression system. More than 15 mg of pure and active PG was obtained from 1 L of shaking‐flask bacteria culture by one‐step nickel affinity chromatography purification. The enzymatic characteristics of the recombinant PG protein were similar to those of native PG. For the deamidation effect of recombinant PG, the deamidation degree (DD) of gliadin reached up to 67% and the solubility increased 84‐fold. Thus, this study provides a practical approach to mass producing active PG proteins and investigates its potential applications on food proteins.

中文翻译：

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使用顺序双表达系统和一步纯化在大肠杆菌中大规模生产活性重组金黄杆菌蛋白水解蛋白谷氨酰胺酶

蛋白质谷氨酰胺酶（Protein glutaminase，PG）是一种特异性催化蛋白质或多肽上谷氨酰胺残基脱酰胺的酶，显着提高了食品蛋白质的溶解性、乳化性和发泡性，在食品工业应用中具有巨大的潜力。PG 主要由蛋白水解金黄色杆菌的野生菌株产生，酶产量低限制了大规模工业应用。在这种情况下，通过评估 PG 前区和成熟结构域之间的不同切割位点插入以及切割位点侧翼的不同接头，已开发出一种大肠杆菌表达和纯化方案来生产活性重组 PG。为了简化生产工作流程，我们开发了一个顺序双表达系统。通过一步镍亲和层析纯化，从 1 L 摇瓶培养物中获得了超过 15 mg 的纯活性 PG。重组PG蛋白的酶学特征与天然PG相似。对于重组PG的脱酰胺作用，麦醇溶蛋白的脱酰胺度（DD）高达67%，溶解度增加了84倍。因此，本研究为大量生产活性 PG 蛋白提供了一种实用的方法，并研究了其在食品蛋白质上的潜在应用。麦醇溶蛋白的脱酰胺度（DD）高达 67%，溶解度增加了 84 倍。因此，本研究为大量生产活性 PG 蛋白提供了一种实用的方法，并研究了其在食品蛋白质上的潜在应用。麦醇溶蛋白的脱酰胺度（DD）高达 67%，溶解度增加了 84 倍。因此，本研究为大量生产活性 PG 蛋白提供了一种实用的方法，并研究了其在食品蛋白质上的潜在应用。