Boonkaew Bootsakorn, Udompaisarn Somsiri, Arthan Dumrongkiet, Somana Jamorn
Siriraj Center for Regenerative Medicine, Siriraj Hospital, Mahidol University, Wanglang Road, Bangkok, 10700, Thailand.
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Paholyothin Road, Pathumthani, 12120, Thailand.
Protein Expr Purif. 2019 Nov;163:105449. doi: 10.1016/j.pep.2019.105449. Epub 2019 Jul 9.
The demand for steviol glycosides, non-caloric sweet components of Stevia rebaudiana Bertoni (stevia) leaves, has increased considerably as a benefit to enhance human health. However, the supply has remained challenging due to limited production, with the lack of a specific steviol glycoside hydrolyzing enzyme. In this study, a novel β-glucosidase (EcBgl) from Enterococcus casseliflavus was cloned and expressed in Escherichia coli. An EcBgl consists of 721 amino acids corresponding to a molecular mass of 79.37 kDa. The EcBgl was purified to homogeneity, followed by enzyme characterization. The enzyme showed optimum pH and temperature at 6.0 and 37 °C, and exhibited the kinetic constants k/K for pNPG and k/K for stevioside of 8583 mMs and 95.41 mMs, respectively. When compared to the stevioside hydrolyzing β-glycosidases previously reported, EcBgl was found to be the most efficient enzyme. EcBgl also rendered hydrolysis of the stevioside to produce rubusoside, a rare steviol glycoside with a pharmaceutical solubilizing property, by cleaving at the glucose moiety. In addition, the enzyme demonstrated substantial resistance against amygdalin, so it served as a potential enzyme in agricultural and pharmaceutical applications.
甜叶菊（Stevia rebaudiana Bertoni）叶中的非热量甜味成分甜菊糖苷的需求因对增进人类健康有益而大幅增加。然而，由于产量有限，且缺乏特定的甜菊糖苷水解酶，其供应仍面临挑战。在本研究中，从格氏肠球菌中克隆出一种新型β-葡萄糖苷酶（EcBgl）并在大肠杆菌中表达。EcBgl由721个氨基酸组成，分子量为79.37 kDa。将EcBgl纯化至同质，随后进行酶特性分析。该酶的最适pH和温度分别为6.0和37°C，对pNPG的动力学常数k/K和对甜菊糖苷的k/K分别为8583 mM/s和95.41 mM/s。与先前报道的甜菊糖苷水解β-糖苷酶相比，EcBgl是最有效的酶。EcBgl还通过切割葡萄糖部分使甜菊糖苷水解生成悬钩子苷，一种具有药物增溶特性的罕见甜菊糖苷。此外，该酶对苦杏仁苷具有显著抗性，因此在农业和制药应用中是一种有潜力的酶。
