Garny Seike, Beeton-Kempen Natasha, Gerber Isak, Verschoor Jan, Jordaan Justin
CSIR Biosciences, Building 20, Meiring Naudé Road, Brummeria, Pretoria, 0001, South Africa; Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria, Private Bag, X20, Hatfield 0028 Pretoria, South Africa.
CSIR Biosciences, Building 20, Meiring Naudé Road, Brummeria, Pretoria, 0001, South Africa.
Enzyme Microb Technol. 2016 Apr;85:71-81. doi: 10.1016/j.enzmictec.2015.10.006. Epub 2015 Oct 23.
The co-immobilization of enzymes on target surfaces facilitates the development of self-contained, multi-enzyme biocatalytic platforms. This generally entails the co-immobilization of an enzyme with catalytic value in combination with another enzyme that performs a complementary function, such as the recycling of a critical cofactor. In this study, we co-immobilized two enzymes from different biological sources for the continuous reduction of nitric oxide, using epoxide- and carboxyl-functionalized hyper-porous microspheres. Successful co-immobilization of a fungal nitric oxide reductase (a member of the cytochrome P450 enzyme family) and a bacterial glucose dehydrogenase was obtained with the carboxyl-functionalized microspheres, with enzyme activity maintenance of 158% for nitric oxide reductase and 104% for glucose dehydrogenase. The optimal stoichiometric ratio of these two enzymes was subsequently determined to enable the two independent chemical reactions to be catalyzed concomitantly, allowing for near-synchronous cofactor conversion rates. This dual-enzyme system provides a novel research tool with potential for in vitro investigations of nitric oxide, and further demonstrates the successful immobilization of a P450 enzyme with potential application towards the immobilization of other cytochrome P450 enzymes.
将酶共固定在目标表面有助于开发独立的多酶生物催化平台。这通常需要将具有催化价值的酶与执行互补功能的另一种酶共固定，例如关键辅因子的循环利用。在本研究中，我们使用环氧基和羧基功能化的超多孔微球，共固定了来自不同生物来源的两种酶，用于连续还原一氧化氮。用羧基功能化微球成功共固定了真菌一氧化氮还原酶（细胞色素P450酶家族的一员）和细菌葡萄糖脱氢酶，一氧化氮还原酶的酶活性维持率为158%，葡萄糖脱氢酶为104%。随后确定了这两种酶的最佳化学计量比，以使两个独立的化学反应能够同时被催化，实现辅因子转化率近乎同步。这种双酶系统为一氧化氮的体外研究提供了一种具有潜力的新型研究工具，并进一步证明了P450酶的成功固定，对其他细胞色素P450酶的固定具有潜在应用价值。
