Torres Rodrigo, Mateo Cesar, Fuentes Manuel, Palomo Jose M, Ortiz Claudia, Fernández-Lafuente Roberto, Guisan Jose M, Tam Andrea, Daminati Moreno
Departmento de Biocatálisis, Instituto de Catalisis, CSIC, Campus Universidad Autónoma, Cantoblanco, Madrid, Spain.
Biotechnol Prog. 2002 Nov-Dec;18(6):1221-6. doi: 10.1021/bp020082q.
Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE-agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers-octamers at low pH value). The optimal enzyme preparation remained fully active after a 15-day incubation at 50 degrees C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 degrees C higher than that of soluble enzyme.
来自酿酒酵母的转化酶已通过离子吸附固定在完全涂覆有聚乙烯亚胺（PEI）的Sepabeads上。该酶强烈吸附在载体上（在能使所有酶从传统阴离子交换剂载体（如DEAE-琼脂糖）上释放的条件下，未发现转化酶解吸）。然而，酶失活后仍可解吸，并且新的新鲜酶可吸附在载体上，而不会对酶负载产生不利影响。这是一种多聚体酶，其最小寡聚化活性状态为二聚体，但在特定的pH和浓度条件下可能形成更大的多聚体。非常有趣的是，结果表明该酶在这种大且灵活的聚合物床上的吸附能够冻结酶的一些不同寡聚结构。因此，我们发现，在某些pH值（pH 8.5）和高酶浓度下固定化的酶（其主要酶结构为四聚体）比在寡聚化不利（低酶浓度下为二聚体）或过高（如低pH值下为六聚体 - 八聚体）的条件下制备的固定化制剂更稳定。在50℃和pH 4.5（标准工业使用条件）下孵育15天后，最佳酶制剂仍保持完全活性，并且其最佳温度比可溶性酶高出约5℃。
