Gupta Shefali, Adlakha Nidhi, Yazdani Syed Shams
Synthetic Biology and Biofuel Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Protein Expr Purif. 2013 Mar;88(1):20-5. doi: 10.1016/j.pep.2012.11.006. Epub 2012 Nov 29.
Escherichia coli is considered one of the most appropriate hosts for the production of recombinant proteins. However, its usage is undermined by its inability to efficiently secrete proteins into the extracellular medium. We selected two cellulolytic enzymes with potential biofuel applications, β-1,4-endoglucanase (Endo5A) and β-1,4-glucosidase (Gluc1C), and determined the genetic and environmental parameters for their optimal secretion into culture medium. Endo5A and Gluc1C were fused with the hyperosmotically inducible periplasmic protein of E. coli, OsmY, and their activities in the extracellular, periplasmic and cytoplasmic fractions were monitored. Most of the endoglucanase activity (0.15 μmol min(-1) ml(-1)) and β-glucosidase activity (2.2 μmol min(-1) ml(-1)) in the extracellular fraction was observed at 16 h post-induction. To reduce the overall cost, we expressed Endo5A and Gluc1C together either via a synthetic operon or through a bifunctional chimeric protein. Both systems efficiently secreted the enzymes, as evident from the functional activities and protein profiles on SDS-PAGE gels. The enzymes secreted via a synthetic operon showed higher activities (0.14 μmol min(-1) ml(-1) for endoglucanase and 2.4 μmol min(-1) ml(-1) for β-glucosidase) as compared to the activities shown by the- bifunctional chimera (0.075 μmol min(-1) ml(-1) for endoglucanase and 2.0 μmol min(-1)ml(-1) for β-glucosidase). The cellulase secretion system developed here has potential for use in the production of lignocellulosic biofuels.
大肠杆菌被认为是生产重组蛋白最合适的宿主之一。然而，由于其无法有效地将蛋白质分泌到细胞外培养基中，其应用受到了限制。我们选择了两种具有潜在生物燃料应用价值的纤维素分解酶，β-1,4-内切葡聚糖酶（Endo5A）和β-1,4-葡糖苷酶（Gluc1C），并确定了它们最佳分泌到培养基中的遗传和环境参数。将Endo5A和Gluc1C与大肠杆菌的高渗诱导周质蛋白OsmY融合，并监测它们在细胞外、周质和细胞质部分的活性。诱导后16小时，在细胞外部分观察到大部分内切葡聚糖酶活性（0.15μmol min⁻¹ ml⁻¹）和β-葡糖苷酶活性（2.2μmol min⁻¹ ml⁻¹）。为了降低总成本，我们通过合成操纵子或双功能嵌合蛋白共同表达Endo5A和Gluc1C。从SDS-PAGE凝胶上的功能活性和蛋白质谱可以明显看出，这两种系统都能有效地分泌这些酶。与双功能嵌合体（内切葡聚糖酶为0.075μmol min⁻¹ ml⁻¹，β-葡糖苷酶为2.0μmol min⁻¹ ml⁻¹）所显示的活性相比，通过合成操纵子分泌的酶显示出更高的活性（内切葡聚糖酶为0.14μmol min⁻¹ ml⁻¹，β-葡糖苷酶为2.4μmol min⁻¹ ml⁻¹）。这里开发的纤维素酶分泌系统有潜力用于木质纤维素生物燃料的生产。
