School of Life Science, East China Normal University, 500 Dongchuan Road, Shanghai, 200241, People's Republic of China.
Curr Microbiol. 2021 May;78(5):1752-1762. doi: 10.1007/s00284-021-02404-0. Epub 2021 Mar 19.
Protein-glutaminase (PG) is a promising protein deaminase. It only hydrolyzes the side chain amido groups of protein-bound to generate ammonia and protein-L-glutamic acid and does not catalyze any other undesirable changes in protein structures. Deamidation of proteins via PG can influence the solubility, emulsification, foaming, and gelation properties of proteins, which are important properties for some food proteins. Therefore, there is great potential for the application of PG in the food industry. PG is derived from Chryseobacterium proteolyticum (C. proteolyticum); however, wild strains are difficult to industrialize because of their low levels of enzyme production. In this article, we studied different strategies for PG expression in B. subtilis. Results showed that PG produced from C. proteolyticum could be successfully secreted in B. subtilis WB800N, and actively secreted in B. subtilis 168(BS168) or DB403 containing a pro-peptide (pro-PG). The secreted PG from B. subtilis WB800N was inactive unless digested by exogenous proteases, such as trypsin, alkaline protease, and neutral protease. However, active PG was secreted by the self-processing of BS168 and DB403. The specific activity of purified PG reached 20.9 U/mg. PG showed maximum activity at pH 5.5, 55 °C and more than 80% of PG activity was retained within a range of pH 3.5-6.5. When Cbz-Gln-Gly was used as the substrate, PG activity was 31.1 ± 0.9 μM min mg. Mg, Ca, and Zn stabilized and even activated PG activity. These strategies concerning PG expression in B. subtilis and the enzymatic properties of PG provide efficient alternatives for PG research and contribute to the industrial-scale production of PG.
蛋白谷氨酰胺酶（PG）是一种很有前途的蛋白脱氨酶。它只水解与蛋白质结合的侧链酰胺基，生成氨和蛋白质 L-谷氨酸，而不会催化蛋白质结构的任何其他不良变化。通过 PG 对蛋白质进行脱酰胺作用，可以影响蛋白质的溶解度、乳化性、起泡性和凝胶性等性质，这些性质对某些食品蛋白质很重要。因此，PG 在食品工业中有很大的应用潜力。PG 来源于 Chryseobacterium proteolyticum（C. proteolyticum）；然而，由于其产酶水平低，野生菌株难以实现工业化。在本文中，我们研究了 PG 在枯草芽孢杆菌中的不同表达策略。结果表明，来自 C. proteolyticum 的 PG 可以在枯草芽孢杆菌 WB800N 中成功分泌，并在含有前肽（pro-PG）的枯草芽孢杆菌 168（BS168）或 DB403 中被主动分泌。除非被外源性蛋白酶（如胰蛋白酶、碱性蛋白酶和中性蛋白酶）消化，否则从枯草芽孢杆菌 WB800N 分泌的 PG 是没有活性的。然而，BS168 和 DB403 可以通过自身加工分泌有活性的 PG。纯化 PG 的比活力达到 20.9 U/mg。PG 在 pH5.5、55°C 时表现出最大活性，在 pH3.5-6.5 范围内保留超过 80%的 PG 活性。当使用 Cbz-Gln-Gly 作为底物时，PG 活性为 31.1±0.9 μM min mg。Mg、Ca 和 Zn 稳定甚至激活 PG 活性。这些枯草芽孢杆菌中 PG 表达的策略和 PG 的酶学性质为 PG 的研究提供了有效的替代方法，并有助于 PG 的工业规模生产。
