Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brasil.
Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brasil.
Braz J Microbiol. 2023 Sep;54(3):1533-1545. doi: 10.1007/s42770-023-01088-x. Epub 2023 Aug 23.
N-Acetyl-glucosaminidases (GlcNAcases) are exoenzymes found in a wide range of living organisms, which have gained great attention in the treatment of disorders related to diabetes, Alzheimer's, Tay-Sachs', and Sandhoff's diseases; the control of phytopathogens; and the synthesis of bioactive GlcNAc-containing products. Aiming at future industrial applications, in this study, GlcNAcase production by marine Aeromonas caviae CHZ306 was enhanced first in shake flasks in terms of medium composition and then in bench-scale stirred-tank bioreactor in terms of physicochemical conditions. Stoichiometric balance between the bioavailability of carbon and nitrogen in the formulated culture medium, as well as the use of additional carbon and nitrogen sources, played a central role in improving the bioprocess, considerably increasing the enzyme productivity. The optimal cultivation medium was composed of colloidal α-chitin, corn steep liquor, peptone A, and mineral salts, in a 5.2 C:N ratio. Optimization of pH, temperature, colloidal α-chitin concentration, and ka conditions further increased GlcNAcase productivity. Under optimized conditions in bioreactor (i.e., 34 °C, pH 8 and ka 55.2 h), GlcNAcase activity achieved 173.4 U.L after 12 h of cultivation, and productivity no less than 14.45 U.L.h corresponding to a 370-fold enhancement compared to basal conditions.
N-乙酰氨基葡萄糖苷酶(GlcNAcases)是广泛存在于各种生物体内的外切酶,在治疗与糖尿病、阿尔茨海默病、泰-萨克斯病和桑德霍夫病相关的疾病、控制植物病原体以及合成具有生物活性的含 GlcNAc 的产品方面引起了极大的关注。鉴于未来的工业应用,本研究首先在摇瓶中针对培养基组成来提高海洋气单胞菌 CHZ306 的 GlcNAcase 生产,然后在台式搅拌罐生物反应器中针对物理化学条件来提高 GlcNAcase 生产。在配方培养基中,碳和氮的生物利用度之间的化学计量平衡,以及额外的碳和氮源的使用,对改善生物过程起着核心作用,极大地提高了酶的生产力。最佳的培养条件由胶体 α-几丁质、玉米浆、蛋白胨 A 和无机盐组成,C:N 比为 5.2。进一步优化 pH 值、温度、胶体 α-几丁质浓度和 ka 值可提高 GlcNAcase 生产率。在生物反应器中的优化条件下(即 34°C、pH8 和 ka 55.2 h),培养 12 h 后 GlcNAcase 活性达到 173.4 U.L,产率不低于 14.45 U.L.h,与基础条件相比提高了 370 倍。