Immobilisation of β-galactosidase within lipid sponge phase: structure, stability and kinetics characterisation
In the formulation of an active enzyme enclosed in a matrix for controlled delivery, it is a challenge to achieve high protein load and to ensure high activity of the protein. For the first time to our knowledge, we report the use of highly swollen lipid sponge (L3) phase for encapsulation of the large active enzyme, β-galactosidase (β-gal, 238 kDa). This enzyme has large relevance for applications in, e.g. the production of lactose free milk products. The formulation consisted of diglycerol monooleate (DGMO), and a mixture of mono-, di- and triglycerides (Capmul GMO-50) stabilised by polysorbate 80 (P80). The advantage of this type of matrix is that it can produced in large scale with a fairly simple and mild process as the system is in practice self-dispersing, yet it is a well-defined internal nano-structure. Minor effects on the sponge phase structure due to the inclusion of the enzyme were observed using small angle x-ray scattering (SAXS). The effect of encapsulation on the enzymatic activity and kinetic characteristics of β-galactosidase activity were also investigated and can be related to the enzyme stability and confinement within the lipid matrix. The encapsulated β-galactosidase maintained its activity for a significantly longer time when compared to the free solution at the same temperature. Differences in particle size and charge of sponge-like nanoparticles (L3-NPs) with and without enzyme were analysed by dynamic light scattering (DLS) and zeta-potential measurements. Moreover, all the initial β-galactosidase was encapsulated within L3-NPs as revealed by size exclusion chromatography.