Molecular Biomaterials, CSIR Biosciences, Meiring Naude Road, Brummeria 0091, South Africa.
Enzyme Microb Technol. 2012 May 10;50(6-7):331-6. doi: 10.1016/j.enzmictec.2012.03.003. Epub 2012 Mar 23.
The use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSyn™ polymer microspheres with varying functional group densities. The successful immobilisation of the enzymes on this new high capacity microsphere technology resulted in the maintenance of activity of ∼40% for GDH and a maximum of 15.4% for NOD. The microsphere variant with highest functional group density of ∼3500 μmol g⁻¹ displayed the highest specific activity for the immobilisation of both enzymes at 33.22 U mg⁻¹ and 6.75 U mg⁻¹ for GDH and NOD with respective loading capacities of 51% (0.51 mg mg⁻¹) and 129% (1.29 mg mg⁻¹). The immobilised GDH further displayed improved activity in the acidic pH range. Both enzymes displayed improved pH and thermal stability with the most pronounced thermal stability for GDH displayed on ReSyn™ A during temperature incubation at 65 °C with a 13.59 fold increase, and NOD with a 2.25-fold improvement at 45 °C on the same microsphere variant. An important finding is the suitability of the microspheres for stabilisation of the multimeric protein GDH.
酶在工业应用中的使用受到其不稳定性、成本以及回收和再利用的困难的限制。固定化是一种已被证明可以缓解生物催化中这些限制的技术。在这里，我们描述了两种与生物催化相关的辅酶再生酶，葡萄糖脱氢酶（GDH）和 NADH 氧化酶（NOD）在醛基功能化的 ReSyn™聚合物微球上的固定化，其功能基团密度不同。成功地将酶固定在这种新的高容量微球技术上，导致 GDH 的活性保持在约 40%，NOD 的最大活性为 15.4%。具有最高功能基团密度的微球变体约为 3500 μmol g⁻¹，对两种酶的固定化表现出最高的比活性，GDH 和 NOD 的比活性分别为 33.22 U mg⁻¹和 6.75 U mg⁻¹，相应的载量分别为 51%（0.51 mg mg⁻¹）和 129%（1.29 mg mg⁻¹）。固定化的 GDH 在酸性 pH 范围内进一步显示出提高的活性。两种酶的 pH 和热稳定性都得到了改善，其中 GDH 在 ReSyn™A 上的热稳定性最为显著，在 65°C 下孵育时，活性增加了 13.59 倍，NOD 在同一微球变体上在 45°C 时，活性提高了 2.25 倍。一个重要的发现是微球适合稳定多聚体蛋白 GDH。
