Dib Iskandar, Nidetzky Bernd
Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria.
BMC Biotechnol. 2008 Sep 17;8:72. doi: 10.1186/1472-6750-8-72.
Immobilization of Trigonopsis variabilis D-amino acid oxidase (TvDAO) on solid support is the key to a reasonably stable performance of this enzyme in the industrial process for the conversion of cephalosporin C as well as in other biocatalytic applications.
To provide a mechanistic basis for the stabilization of the carrier-bound oxidase we analyzed the stabilizing effects of immobilization in TvDAO exposed to the stress of elevated temperature and operational conditions. Two different strategies of immobilization were used: multi-point covalent binding to epoxy-activated Sepabeads EC-EP; and non-covalent oriented immobilization of the enzyme through affinity of its N-terminal Strep-tag to Strep-Tactin coated on insoluble particles. At 50 degrees C, the oriented immobilizate was not stabilized as compared to the free enzyme. The structure of TvDAO was stabilized via covalent attachment to Sepabeads EC-EP but concomitantly, binding of the FAD cofactor was weakened. FAD release from the enzyme into solution markedly reduced the positive effect of immobilization on the overall stability of TvDAO. Under conditions of substrate conversion in a bubble-aerated stirred tank reactor, both immobilization techniques as well as the addition of the surfactant Pluronic F-68 stabilized TvDAO by protecting the enzyme from the deleterious effect of gas-liquid interfaces. Immobilization of TvDAO on Sepabeads EC-EP however stabilized the enzyme beyond this effect and led to a biocatalyst that could be re-used in multiple cycles of substrate conversion.
Multi-point covalent attachment of TvDAO on an isoluble porous carrier provides stabilization against the denaturing effects of high temperature and exposure to a gas-liquid interface. Improvement of binding of the FAD cofactor, probably by using methods of protein engineering, would further enhance the stability of the immobilized enzyme.
将可变三角酵母D-氨基酸氧化酶（TvDAO）固定在固体载体上是该酶在头孢菌素C转化的工业过程以及其他生物催化应用中实现合理稳定性能的关键。
为了为固定化载体结合氧化酶的稳定化提供机理基础，我们分析了TvDAO在暴露于高温和操作条件压力下的固定化稳定作用。使用了两种不同的固定化策略：与环氧活化的Sepabeads EC-EP进行多点共价结合；以及通过其N端链霉亲和标签与涂覆在不溶性颗粒上的链霉抗生物素蛋白的亲和力对酶进行非共价定向固定。在50℃时，与游离酶相比，定向固定化产物未得到稳定。TvDAO的结构通过与Sepabeads EC-EP的共价连接而稳定，但同时，FAD辅因子的结合减弱。FAD从酶中释放到溶液中显著降低了固定化对TvDAO整体稳定性的积极影响。在鼓泡搅拌釜反应器中的底物转化条件下，两种固定化技术以及表面活性剂普朗尼克F-68的添加都通过保护酶免受气液界面的有害影响而稳定了TvDAO。然而，将TvDAO固定在Sepabeads EC-EP上使酶的稳定效果超出了这一范围，并产生了一种可在多个底物转化循环中重复使用的生物催化剂。
TvDAO在不溶性多孔载体上的多点共价连接提供了针对高温变性作用和暴露于气液界面的稳定性。可能通过蛋白质工程方法改善FAD辅因子的结合，将进一步提高固定化酶的稳定性。
