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Reactivation of lipases by the unfolding and refolding of covalently immobilized biocatalysts

机译：通过共价固定的生物催化剂的折叠和重折叠来活化脂肪酶

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摘要

Lipases from Candida antarctica (isoform B) (CALB) and Thermomyces lanuginosus (TLL) have been immobilized either covalently or by interfacial activation versus an octyl support, followed by covalent attachment by glyoxyl groups using octyl-glyoxyl agarose beads (OCGLX). These biocatalysts have been submitted to successive cycles of unfolding by incubation in 9 M guanidine and refolding by incubation in aqueous 100 mM phosphate buffer at pH 7, before and after total inactivation in the presence of organic solvents. The four preparations have been reactivated to some extent using this strategy, but the results depended on the method of preparation. Glyoxyl-immobilized CALB may recover 100% of its activity versus p-nitrophenyl butyrate, but after solvent inactivation the recovery of activity was reduced to 95%. The pure covalent TLL preparation recovered around 80% of its activity, either before or after solvent inactivation. Both enzymes showed less recovery of activity using OCGLX, as might be expected from the hydrophobic nature of the supporting groups (60% for CALB and 45% for TLL). In addition, using enzymes previously inactivated by solvent, recovery decreased by 5-10%. These values were maintained through three successive cycles. However, using R-and S-methyl mandelate, it was clear that recovery of activity decreased with further reactivation cycles. Taken as a whole, the unfolding and refolding effect may be used to recover a degree of enzyme activity. This is relevant in terms of application, as it may allow the enzyme preparations to be used for a longer period. However, to reach a similar enzyme structure in each reactivation cycle, it will be necessary to undertake further studies involving the use of other supports in order to improve the unfolding and refolding steps.
机译：来自南极假丝酵母（异构体B）（CALB）和羊毛嗜热霉（TLL）的脂肪酶已通过共价固定或通过界面活化相对于辛基支持物进行固定，然后使用辛基-乙醛酸琼脂糖珠（OCGLX）通过乙醛基进行共价连接。这些生物催化剂在有机溶剂存在下完全灭活之前和之后，通过在9 M胍中温育和在100 mM磷酸盐水溶液pH 7中温育重折叠进行了连续的连续循环。使用此策略可以在某种程度上重新激活这四种制剂，但结果取决于制备方法。相对于对硝基苯基丁酸酯，乙二醛固定化的CALB可以恢复其活性的100％，但是在溶剂失活后，活性的恢复率降低到了95％。在溶剂灭活之前或之后，纯共价TLL制剂的活性恢复了约80％。两种酶均显示出使用OCGLX的活性恢复较少，这可能是由支持基团的疏水性所预期的（CALB为60％，TLL为45％）。另外，使用先前被溶剂灭活的酶，回收率降低了5-10％。这些值通过三个连续的周期保持。然而，很明显，使用R-和S-扁桃酸甲酯，活性的恢复随着进一步的再活化循环而降低。总体而言，解折叠和再折叠作用可用于恢复一定程度的酶活性。就应用而言，这是相关的，因为它可以使酶制剂更长时间地使用。然而，为了在每个活化周期中达到相似的酶结构，有必要进行进一步的研究，包括使用其他支持物，以改善解折叠和重折叠步骤。

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