Boric acid, B(OH)3 (or its salt Borax) is known to stabilize serine proteases by inhibition of the proteolytic activity and boric acid in combination with polyols is the most commonly used stabilization system in liquid detergents. The concentration of boric acid in the liquid detergent needed to obtain stabilization is typically 1-3% (w/w). Enzymes are typically used in concentrations below 2%, and due to the solubility of boric acid/borax it is thus not possible to supply an enzyme product with a built-in boric acid stabilization system that will also give stability in the final detergent. The design of such products must thus be based on other stabilization technologies.
Three possible stabilization technologies were identified: Designing more stable enzymes e.g. by genetically engineering Micro-encapsulation of enzymes Designing more efficient inhibitors From an evaluation of pros and cons the last one was pursued.
A vast number of different protease inhibitors exist. However, due to similarity with the existing boric acid system it was decided to focus the screening around boronic acids R-B(OH)2, where R is an organic moiety. A partner with more knowledge of boronic acid chemistry was included in the development.
Possible candidates were identified using several techniques: Testing whatever readily available Production of trial-and-error candidates Computer modelling (docking of inhibitors candidates into enzyme molecules) Systematically pursuing positive leads
The screening tools were chosen so it balanced with the number of inhibitor variants we expected to produce in this case in the magnitude of 10 per week. To minimize the risk of false negative results two screening systems were used: Studies of enzyme kinetics in buffer Storage stability in a model detergent Candidates with a positive result in both assays were chosen for further studies and development. The two assays showed a reasonable but not complete correlation.
4-formyl phenyl boronic acid (4-FPBA) was found to be the most promising candidate and the intensive testing, development of the enzyme/inhibitor formulation and toxicological studies were successful. The most time consuming part of the project turned out to be development and up-scaling of the inhibitor to an economical industrial scale. The complexity of this part was probably underestimated initially in the project.
Novozymes now have several enzyme products on the market with built-in stabilization system based on 4-FPBA inhibitor.