Uhoraningoga Albert, Kinsella Gemma K, Frias Jesus M, Henehan Gary T, Ryan Barry J
School of Food Science and Environmental Health, College of Sciences and Health, Technological University Dublin, Dublin D07 ADY7, Ireland.
Bioengineering (Basel). 2019 Jul 18;6(3):61. doi: 10.3390/bioengineering6030061.
β-glucosidases are a class of enzyme that are widely distributed in the living world, with examples noted in plants, fungi, animals and bacteria. They offer both hydrolysis and synthesis capacity for a wide range of biotechnological processes. However, the availability of native, or the production of recombinant β-glucosidases, is currently a bottleneck in the widespread industrial application of this enzyme. In this present work, the production of recombinant β-glucosidase from was optimised using a Design of Experiments strategy, comprising a two-stage, multi-model design. Three screening models were comparatively employed: Fractional Factorial, Plackett-Burman and Definitive Screening Design. Four variables (temperature, incubation time, tryptone, and OD) were experimentally identified as having statistically significant effects on the production of recombinant β-glucosidase in BL21 (DE3). The four most influential variables were subsequently used to optimise recombinant β-glucosidase production, employing Central Composite Design under Response Surface Methodology. Optimal levels were identified as: OD, 0.55; temperature, 26 °C; incubation time, 12 h; and tryptone, 15 g/L. This yielded a 2.62-fold increase in recombinant β-glucosidase production, in comparison to the pre-optimised process. Affinity chromatography resulted in homogeneous, purified β-glucosidase that was characterised in terms of pH stability, metal ion compatibility and kinetic rates for -nitrophenyl-β-D-glucopyranoside (NPG) and cellobiose catalysis.
β-葡萄糖苷酶是一类广泛分布于生物界的酶，在植物、真菌、动物和细菌中均有发现。它们在广泛的生物技术过程中兼具水解和合成能力。然而，天然β-葡萄糖苷酶的可得性或重组β-葡萄糖苷酶的生产，目前是该酶广泛工业应用的一个瓶颈。在本研究中，采用实验设计策略对[具体来源]重组β-葡萄糖苷酶的生产进行了优化，该策略包括两阶段、多模型设计。比较采用了三种筛选模型：分数析因设计、Plackett-Burman设计和确定性筛选设计。通过实验确定了四个变量（温度、培养时间、胰蛋白胨和光密度）对BL21(DE3)中重组β-葡萄糖苷酶的生产具有统计学显著影响。随后，利用响应面法下的中心复合设计，将这四个最具影响力的变量用于优化重组β-葡萄糖苷酶的生产。确定的最佳水平为：光密度0.55；温度26℃；培养时间12小时；胰蛋白胨15 g/L。与优化前的工艺相比，重组β-葡萄糖苷酶的产量提高了2.62倍。亲和层析得到了均一、纯化的β-葡萄糖苷酶，并对其pH稳定性、金属离子兼容性以及对对硝基苯基-β-D-吡喃葡萄糖苷（NPG）和纤维二糖催化的动力学速率进行了表征。
