Controlling enzyme crystals' features in a membrane crystallization apparatus

To grow protein crystals has been so far recognized as a not easy task, due to the high level of complexity of bio-macromolecules. Furthermore, once crystals have been produced, their characteristics, determined heavily from the crystallization kinetic, may not be well matching the necessary requirements with respect to the intended uses. However, an increasingly consistent input for proteins and more specifically enzymes crystallizations derives from the wider use of cross linked enzyme crystals (CLECs) in biotechnological areas. Such materials, with growing industrial applications, generally demonstrated enhanced stability against denaturation by heat, organic solvents and proteolysis, and, depending on some parameters, exhibited no loss or even increase in enzymatic activity in the cross-linked crystalline formulation with respect to the corresponding solution state. This behaviour is heavily dependent by the morphological and structural characteristics of CLECs, in particular for chemical applications that require biocatalyst recycle. To produce enzyme crystals by an opportune technique that allows to control the final properties of the products, would be thus an essential improvement in the overall field of life sciences. In this respect, membrane crystallization has been recently proposed as a new technique for producing protein crystals with enhanced crystallization kinetic, by utilising the polymeric membrane surface as a promoter of heterogeneous crystallization. Preliminary important results obtained using lysozyme as model protein, in both static and forced solution configuration, have been followed by further investigations on other enzymes, such as trypsins. In the presented work are described the last developments regarding the use of membrane crystallization for better understand and control, by acting on the operative parameters involved in the process, the crystallization kinetic of enzymes for producing crystals with adequate characteristics, in terms of size, size distribution, internal quality, depending on the specific application required, which may be to produce single crystals, for structural investigations, or crystalline materials as the first step in the formulation of CLECs.