Glyoxyl-Disulfide Agarose: A Tailor-Made Support for Site-Directed Rigidification of Proteins

A new strategy has been developed for site-directed immobilization/rigidification of genetically modified enzymes through multipoint covalent attachment on bifunctional disulfide-glyoxyl supports. Here the mechanism is described as a two-step immobilization/rigidification protocol where the enzyme is directly immobilized by thiol-disulfide exchange between the β-thiol of the single genetically introduced cysteine and the few disulfide groups presented on the support surface (3 μmol/g). Afterward, the enzyme is uniquely rigidified by multipoint covalent attachment (MCA) between the lysine residues in the vicinity of the introduced cysteine and the many glyoxyl groups (220 μmol/g) on the support surface. Both site-directed immobilization and rigidification have been possible only on these novel bifunctional supports. In fact, this technology has made possible to elucidate the protein regions where rigidification by MCA promoted higher protein stabilizations. Hence, rigidification of vicinity of position 333 from lipase 2 from Geobacillus thermocatenulatus (BTL2) promoted a stabilization factor of 33 regarding the unipunctual site-directed immobilized derivative. In the same context, rigidification of penicillin G acylase from E. coli (PGA) through position β201 resulted in a stabilization factor of 1069. Remarkably, when PGA was site-directed rigidified through that position, it presented a half-life time of 140 h under 60% (v/v) of dioxane and 4 °C, meaning a derivative eight times more stable than the PGA randomly immobilized on glyoxyl-disulfide agarose. Herein we have opened a new scenario to optimize the stabilization of proteins via multipoint covalent immobilization, which may represent a breakthrough in tailor-made tridimensional rigidification of proteins.

本研究开发了一种新型的策略，通过在双功能二硫键-甘露醇基载体上的多点共价连接，实现了对基因改良酶的定向固定/刚性化。该机制被描述为一种两步固定/刚性化协议，其中酶通过引入的单个半胱氨酸的β-巯基与载体表面存在的少量二硫键（3 μmol/g）之间的巯基-二硫键交换而直接固定。随后，酶通过在引入的半胱氨酸附近的赖氨酸残基与载体表面众多的甘露醇基团（220 μmol/g）之间的多点共价连接（MCA）来实现独特的刚性化。仅在新型双功能载体上才实现了定向固定和刚性化。实际上，这项技术使得阐明通过MCA刚性化促进的蛋白质区域，从而提高了蛋白质的稳定性成为可能。因此，来自地芽孢杆菌热链菌（Geobacillus thermocatenulatus）的脂肪酶2（BTL2）在位置333附近的刚性化，相对于单点定向固定的衍生物，其稳定性因子提高了33。在同一背景下，通过位置β201对青霉素G酰化酶（PGA）的刚性化导致其稳定性因子达到1069。值得注意的是，当PGA通过该位置进行定向刚性化时，在60%（体积比）的二氧烷和4°C下，其半衰期为140小时，这意味着其衍生物的稳定性是随机固定在甘露醇-二硫键琼脂糖上的PGA的八倍。本研究开辟了通过多点共价固定优化蛋白质稳定性的新途径，这可能代表着在蛋白质定制三维刚性化方面的突破。