Mei Wending, Wang Lu, Zang Ying, Zheng Zhaojuan, Ouyang Jia
College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, People's Republic of China.
College of Forestry, Nanjing Forestry University, Nanjing, 210037, People's Republic of China.
BMC Biotechnol. 2016 Jun 30;16(1):55. doi: 10.1186/s12896-016-0286-5.
L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed.
The coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn(2+) at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (k cat/K m) for L-arabinose and D-galactose were 8.7 mM(-1) min(-1) and 1.0 mM(-1) min(-1) respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L(-1) and 250 g L(-1) D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h).
In this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn(2+). Its substrate specificity was understood deeply by carrying out molecular modelling and docking studies. When the recombinant E. coli expressing the AI was used as a biocatalyst, D-tagatose could be produced efficiently in a simple one-pot biotransformation system.
L-阿拉伯糖异构酶（AI）是将D-半乳糖生物转化为D-塔格糖的关键催化剂。在以往的报道中，嗜热细菌菌株来源的AI已得到广泛研究，但高温下的褐变反应和副产物形成限制了它们的应用。相比之下，嗜温芽孢杆菌菌株来源的AI具有一些不同的特性，包括较低的最适温度和对金属辅因子的较低需求。这些特性将有利于开发更节能、更安全的生产工艺。然而，关于芽孢杆菌AI在D-塔格糖生产中的动力学和反应特性的相关数据仍然不足。因此，为了支持这些AI的进一步应用，需要对芽孢杆菌AI进行全面表征。
凝结芽孢杆菌NL01 AI（BCAI）的编码基因（1422 bp）被克隆并在大肠杆菌BL21（DE3）菌株中过表达。酶学性质测试表明，BCAI的最适温度和pH分别为60℃和7.5。粗纯化的BCAI在没有外源金属离子的情况下最初表现出高活性，并且在70℃至90℃的温度下，其热稳定性在低浓度（0.5 mM）的Mn(2+)中没有变化。除此之外，BCAI对L-阿拉伯糖和D-半乳糖的催化效率（k cat/K m）分别为8.7 mM(-1) min(-1)和1.0 mM(-1) min(-1)。在最佳条件下，含有BCAI的重组大肠杆菌细胞可以将150 g L(-1)和250 g L(-1)的D-半乳糖转化为D-塔格糖，转化率分别为32%（32小时）和27%（48小时），具有吸引力。
在本研究中，一种来自凝结芽孢杆菌NL01的新型AI被克隆、纯化和表征。与其他报道的AI相比，这种AI在更宽的温度范围内能保持较高比例的活性，并且对诸如Mn(2+)等金属辅因子的依赖性较小。通过进行分子建模和对接研究，深入了解了其底物特异性。当表达该AI的重组大肠杆菌用作生物催化剂时，D-塔格糖可以在简单的一锅生物转化系统中高效生产。
Zotero 插件
