Parkin K, Hultin H O
Biotechnol Bioeng. 1979 Jun;21(6):939-53. doi: 10.1002/bit.260210603.
Optimal conditions with respect to pH, concentration of glutaraldehyde and enzyme, and order of addition of enzyme and crosslinking reagent were established for the immobilization of hog kidney D-amino acid oxidase to an attapulgite support. Yields of 40 to 70% were generally attained although when low concentrations of enzyme were used yields were consistently greater than 100%. It is suggested that this is due to a dimer leads to monomer shift at low protein concentrations. The stability of soluble D-amino acid oxidase was dependent on the buffer in which it was stored (pyrophosphate-phosphate greater than borate greater than Tris). Stability of immobilized enzyme was less than soluble in pyrophosphate-phosphate buffer, but storage in the presence of FAD improved stability. In addition, treatment of stored, immobilized enzyme with FAD before assay restored some of its activity. The immobilized D-amino acid oxidase was less stable to heat (50 degrees C) than the soluble enzyme from pH 6 to 8 but was more stable above and below these values. Apparent Km values for D-alanine, D-valine, and D-tryptophan decreased for the immobilized enzyme compared to the soluble.
确定了将猪肾D-氨基酸氧化酶固定在凹凸棒土载体上时，关于pH、戊二醛和酶的浓度以及酶和交联剂添加顺序的最佳条件。尽管使用低浓度酶时产率始终大于100%，但通常产率可达40%至70%。据推测，这是由于在低蛋白浓度下二聚体向单体的转变所致。可溶性D-氨基酸氧化酶的稳定性取决于其储存所用的缓冲液（焦磷酸-磷酸盐大于硼酸盐大于Tris）。固定化酶在焦磷酸-磷酸盐缓冲液中的稳定性低于可溶性酶，但在FAD存在下储存可提高稳定性。此外，在测定前用FAD处理储存的固定化酶可恢复其部分活性。固定化D-氨基酸氧化酶在50℃下，从pH 6至8比可溶性酶对热更不稳定，但在这些值以上和以下则更稳定。与可溶性酶相比，固定化酶对D-丙氨酸、D-缬氨酸和D-色氨酸的表观Km值降低。
