Production of Cross-Linked Lipase Crystals at a Preparative Scale

Abstract

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 p -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.

Solid-state NMR – a complementary technique for protein framework characterization

“…Protein crystals, which for decades have enabled advances in biomedical research, are currently in development as reaction vessels and templating devices with potential therapeutic applications. 1,2 Such materials are attractive as they are sustainable, biocompatible, programmable (from primary structure to crystalline assembly), and possess highly selective recognition and catalytic activities. Considerable effort is being invested in porous protein crystals, which possess large well-defined pores that permit the uptake and release of substrate/product cargo, enabling, for example, controlled drug delivery.…”

Magnetite Mineralization inside Cross-Linked Protein Crystals

“…Although protein crystals are mechanically unstable, cross-linking these crystals can improve their stability and maintain their shape and catalytic activity or being used as heterogeneous catalysts even after being dry for preservation. − In fact, cross-linked protein crystals (CLPCs) have been explored for the formation of new inorganic or hybrid materials by a process that implies metal or chemical coordination with the amino acids exposed to the solvent channels and a subsequent reduction or precipitation of the particles . This technique has been used to obtain well-ordered arrays of metallic nanoparticles of Pd/Pt, Ag, − Au, ,− Co/Pt, and CdS quantum dots, giving rise to composite protein crystals with new catalytic and optical properties.…”

Performance of cross‐linked enzyme crystals of engineered halohydrin dehalogenase HheG in different chemical reactor systems

“…Here, enzymes can be cross-linked as soluble (Ashjari et al, 2020) or lyophilized enzyme (Akkas et al, 2020), as well as in the form of enzyme aggregates (Sheldon, 2007) or enzyme crystals (Häring & Schreier, 1999). Cross-linked enzyme crystals (CLECs) and cross-linked enzyme aggregates (CLEAs) have been investigated and widely used in biocatalytic reactions so far (Fernández-Penas et al, 2021;Mahmod et al, 2016;Perveen et al, 2022;Rehman et al, 2016;St. Clair & Navia, 1992;Vaghjiani et al, 2000).…”