Narayanan Ravindrabharathi, Zhu Guangyu, Wang Ping
Department of Chemical Engineering, The University of Akron, Akron, OH 44325-3906, USA.
J Biotechnol. 2007 Jan 30;128(1):86-92. doi: 10.1016/j.jbiotec.2006.08.017. Epub 2006 Dec 8.
The stability of an interface-binding chloroperoxidase (CPO) against the deactivation effect of H(2)O(2) was examined. Native CPO was conjugated with polystyrene and thus self-assembled at the water-oil interface. Although the interface-assembled CPO showed improved stability as compared to native CPO, enzyme deactivation as a result of the side effect of H(2)O(2), still limits the overall productivity of the enzyme. Two approaches to further improve the stability of CPO were examined in this work. In one approach, several stabilizers including poly(ethylene glycol) (PEG), PEI, glycerol, sugars and sucrose monododecanoate were used; while in a second approach, in situ generation of hydrogen peroxide (H(2)O(2)) by using glucose oxidase (GOx) was applied. PEG was found exceptional in that it increased both the operational and storage stability of CPO. The best improvement of enzyme productivity was obtained with addition of PEG which led to an increase of 57% for interface-bound CPO and 33% for native CPO. One interesting observation with PEI is that it enhanced the storage stability against H(2)O(2) deactivation, but did not affect the enzyme's operational stability. On the other hand, glucose enhanced the operational stability by two folds, but exhibited no significant effect on storage stability. It was also found that the extended operational lifetime of CPO with in situ generation of H(2)O(2) by GOx was a result that combines the stabilizing effect of glucose and lowered concentration of H(2)O(2). Interestingly, the addition of stabilizers could improve the enantioselectivity of CPO by as much as 10%.
研究了界面结合氯过氧化物酶（CPO）对H₂O₂失活作用的稳定性。天然CPO与聚苯乙烯共轭，从而在水-油界面自组装。尽管与天然CPO相比，界面组装的CPO稳定性有所提高，但由于H₂O₂的副作用导致的酶失活仍然限制了酶的整体生产率。在这项工作中研究了两种进一步提高CPO稳定性的方法。一种方法是使用几种稳定剂，包括聚乙二醇（PEG）、聚乙烯亚胺（PEI）、甘油、糖类和蔗糖单十二烷酸酯；而在第二种方法中，应用葡萄糖氧化酶（GOx）原位生成过氧化氢（H₂O₂）。发现PEG非常特殊，因为它提高了CPO的操作稳定性和储存稳定性。添加PEG可使酶生产率得到最佳提高，界面结合的CPO提高了57%，天然CPO提高了33%。关于PEI的一个有趣观察结果是，它增强了对H₂O₂失活的储存稳定性，但不影响酶的操作稳定性。另一方面，葡萄糖使操作稳定性提高了两倍，但对储存稳定性没有显著影响。还发现，通过GOx原位生成H₂O₂可延长CPO的操作寿命，这是葡萄糖的稳定作用和降低H₂O₂浓度共同作用的结果。有趣的是，添加稳定剂可使CPO的对映选择性提高多达10%。
