The Mechanisms of Protein Stabilisation

Applied Enzyme Technology Ltd. has been pioneering research into understanding the mechanisms of protein stabilization in order to more efficiently predict the stabilisation formulation for a given protein in a specific medium. Our platform technology is based upon the unique combination of specific polyalcohol and polyelectrolyte molecules.

Since AET was launched in 1994, the company has now identified a "next generation" of stabiliser molecules which give further stability to proteins and more versatility for the wider application of stabiliser formulations in industry. This is the basis of our current business. AET has launched the production of a protein stabilization kit using this “next generation” of stabiliser formulations which stabilises of the majority of enzymes and proteins identified of interest in industrial processes both in solution and in the dry state. Examples of applications the environmental industries, the house-hold product and hygiene industries, the diagnostics industries and pharmaceutical industries.

Polyalcohols

The polyalcohols include sugars, sugar alcohols and other more exotic substituted sugar molecules. These polyhydric molecules modify the water environment surrounding a protein competing for and replacing the free water, thus modifying the hydration shell of the protein in question. This modified hydration shell confers protection to the protein while maintaining 3D structure and biological activity.

The storage of biological materials in the presence of these polyalcohol molecules allows storage in solution and in the dehydrated state with vastly decreased levels of biological activity with time.

Polyelectrolytes

The polyelectrolytes include numerous polymers of varying size charge and structure. The interactions between proteins and polymers are electrostatic. Evidence for this is now widespread. These form large protein-polyelectrolyte complexes which retain full biological activity

At AET we have the ability to calculate the stoichiometry of protein/polymer binding which we have determined as critical for maintaining enzyme activity in stabiliser formulations

Polyalcohol/polyelectrolyte combinations

AET’s platform technology is based on the combination of these stabiliser compounds. We possess many years of results that prove the combination of polyalcohols with polyelectrolytes leads to a synergistic stabilisation effect. The reason for this synergism is a matter of debate. At AET we have been examining how certain interactions are generated between stabiliser molecules and proteins, how this effects their overall stability and how this can enable us to more accurately predict an optimal stabiliser formulation. Our research shows that polyelectrolyte molecules bind multiple protein molecules depending on the size of the polyelectrolyte involved.(See theroretical model below). Indeed many small polyelectrolyte molecules will bind to a large protein molecule. Furthermore, the smaller polyalcohol molecules will fit within the matrix generated by the protein/polyelectrolyte complex.

Protein polyelectrolyte complexes

Protein-Polyelectrolyte Complexes

The addition of polyelectrolytes to solutions of proteins promotes the formation of soluble protein - polyelectrolyte complexes by electrostatic interaction. Polyhydroxyl compounds are then able to penetrate the structure more effectively leading to stabilisation.

From the research carried out at AET, it is apparent that the ratios of polyelectrolyte to polyalcohol are extremely important in the overall stabilisation of proteins. Obviously the buffer type, pH, ionic strength, concentration and ratio of stabilisers to protein/enzyme all play crucial roles in protein stabilisation, both in the dry state and in solution. This is the basis of any feasibility study we carry out for a client.