Betancor Lorena, Hidalgo Aurelio, Fernández-Lorente Gloria, Mateo Cesar, Rodríguez Verónica, Fuentes Manuel, López-Gallego Fernando, Fernández-Lafuente Roberto, Guisan José M
Laboratorio de Tecnología Enzimática, Departamento de Biocatálisis, ICP-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.
Biotechnol Prog. 2003 May-Jun;19(3):784-8. doi: 10.1021/bp025761f.
An evaluation of the stability of several forms (including soluble and two immobilized preparations) of d-amino acid oxidases from Trigonopsis variabilis (TvDAAO) and Rhodotorula gracilis (RgDAAO) is presented here. Initially, both soluble enzymes become inactivated via subunit dissociation, and the most thermostable enzyme seemed to be TvDAAO, which was 3-4 times more stable than RgDAAO at a protein concentration of 30 microg/mL. Immobilization on poorly activated supports was unable to stabilize the enzyme, while highly activated supports improved the enzyme stability. Better results were obtained when using highly activated glyoxyl agarose supports than when glutaraldehyde was used. Thus, multisubunit immobilization on highly activated glyoxyl agarose dramatically improved the stability of RgDAAO (by ca. 15,000-fold) while only marginally improving the stability of TvDAAO (by 15-20-fold), at a protein concentration of 6.7 microg/mL. Therefore, the optimal immobilized RgDAAO was much more stable than the optimal immobilized TvDAAO at this enzyme concentration. The lower stabilization effect on TvDAAO was associated with the inactivation of this enzyme by FAD dissociation that was not prevented by immobilization. Finally, nonstabilized RgDAAO was marginally more stable in the presence of H(2)O(2) than TvDAAO, but after stabilization by multisubunit immobilization, its stability became 10 times higher than that of TvDAAO. Therefore, the most stable DAAO preparation and the optimal choice for an industrial application seems to be RgDAAO immobilized on glyoxyl agarose.
本文对来自可变三角酵母（TvDAAO）和纤细红酵母（RgDAAO）的几种形式（包括可溶性和两种固定化制剂）的d -氨基酸氧化酶的稳定性进行了评估。最初，两种可溶性酶都通过亚基解离而失活，最耐热的酶似乎是TvDAAO，在蛋白质浓度为30微克/毫升时，其稳定性比RgDAAO高3 - 4倍。固定在活化程度低的载体上无法使酶稳定，而活化程度高的载体则提高了酶的稳定性。使用高度活化的乙醛酸琼脂糖载体比使用戊二醛能获得更好的结果。因此，在蛋白质浓度为6.7微克/毫升时，多亚基固定在高度活化的乙醛酸琼脂糖上可显著提高RgDAAO的稳定性（约15000倍），而对TvDAAO稳定性的提高幅度较小（15 - 20倍）。因此，在该酶浓度下，最佳固定化的RgDAAO比最佳固定化的TvDAAO稳定得多。对TvDAAO较低的稳定化效果与该酶因黄素腺嘌呤二核苷酸（FAD）解离而失活有关，固定化并不能阻止这种解离。最后，未稳定化的RgDAAO在过氧化氢存在下比TvDAAO略稳定，但通过多亚基固定化稳定后，其稳定性比TvDAAO高10倍。因此，最稳定的DAAO制剂以及工业应用的最佳选择似乎是固定在乙醛酸琼脂糖上的RgDAAO。
