Fernández-Penas Raquel, Verdugo-Escamilla Cristóbal, Martínez-Rodríguez Sergio, Gavira José A
Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la Tierra, Consejo Superior de Investigaciones Científicas-Universidad de Granada, Avenida de las Palmeras 4, Armilla, 18100 Granada, Spain.
Departamento de Bioquímica y Biología Molecular III e Inmunología, Universidad de Granada, Avenida de la Investigación 11, 18071 Granada, Spain.
Cryst Growth Des. 2021 Mar 3;21(3):1698-1707. doi: 10.1021/acs.cgd.0c01608. Epub 2021 Feb 17.
The autoimmobilization of enzymes via cross-linked enzyme crystals (CLECs) has regained interest in recent years, boosted by the extensive knowledge gained in protein crystallization, the decrease of cost and laboriousness of the process, and the development of potential applications. In this work, we present the crystallization and preparative-scale production of reinforced cross-linked lipase crystals (RCLLCs) using a commercial detergent additive as a raw material. Bulk crystallization was carried out in 500 mL of agarose media using the batch technique. Agarose facilitates the homogeneous production of crystals, their cross-linking treatment, and their extraction. RCLLCs were active in an aqueous solution and in hexane, as shown by the hydrolysis of -nitrophenol butyrate and α-methylbenzyl acetate, respectively. RCLLCs presented both high thermal and robust operational stability, allowing the preparation of a packed-bed chromatographic column to work in a continuous flow. Finally, we determined the three-dimensional (3D) models of this commercial lipase crystallized with and without phosphate at 2.0 and 1.7 Å resolutions, respectively.
近年来，通过交联酶晶体（CLEC）实现的酶自身固定化重新引起了人们的关注，这得益于在蛋白质结晶方面积累的丰富知识、该过程成本和繁琐程度的降低以及潜在应用的发展。在这项工作中，我们展示了以一种商业洗涤剂添加剂为原料制备强化交联脂肪酶晶体（RCLLC）及其规模化生产的过程。采用分批技术在500 mL琼脂糖培养基中进行大量结晶。琼脂糖有助于晶体的均匀生成、交联处理及其提取。分别通过对丁酸对硝基苯酚酯和α-甲基苄基乙酸酯的水解表明，RCLLC在水溶液和己烷中均具有活性。RCLLC具有高热稳定性和强大的操作稳定性，能够制备用于连续流动的填充床色谱柱。最后，我们分别以2.0 Å和1.7 Å的分辨率确定了该商业脂肪酶在有磷酸盐和无磷酸盐情况下结晶的三维（3D）模型。
