Creative Biolabs offers customers a robust platform for enhancing enzyme stability. This platform has been successfully used to increase the thermo-stability and half-life of an enzyme.
Enhancement of enzyme stability means to improve the enzyme stability alongside the specificity and activity. Stability is one of the most desirable properties of an enzyme to be improved, the enhancement of which has been widely practiced both industrially and pharmacologically. Examples of such enhancement can be the sustained enzymatic activity over a greater range of temperatures and decreased susceptibility to cleavage by proteases. Many different approaches have been applied to enhance the stability, but few of them have been successful. We have developed a working platform that can increase the thermo-stability and half-life of a given enzyme. This is achieved by the various techniques of the platform, such as the terminal truncation, random mutagenesis and fragmentation, recombination, elongation, and finally selection at physiological temperatures.
Figure 1. Scheme of truncation–optimization–elongation.
Hydrophobic interaction and hydrogen bonding are the most prevalent in proteins. The hydrophobic effect is considered to be the major driving force for the folding of globular proteins, and results in the burial of the hydrophobic residues in the core of the protein. Another important factor that stabilize the unfolded state is the conformational entropy. Salt bridges or ion-pairs are a special form of particularly strong hydrogen bonds made up of the interaction between two charged residues. The contribution of salt bridges to protein stability is a somewhat contentious issue in the literature. Perhaps at higher temperatures salt bridges make more of a contribution to stability. Disulphide bonds are formed by the oxidation of two cysteine residues to form a covalent sulphur-sulphur bond, which can be intra- or inter- molecular bridges. Though its enthalpy is very high, this bond is present in both the folded and the unfolded state, thus its enthalpic contribution to the free energy difference is negligible. About 60% of the aromatic side chains (Phe, Tyr, and Trp), found in proteins are involved in aromatic pairings. Studies with model compounds suggest that the optimal geometry is perpendicular, such that the partially positively charged hydrogens on the edge of one ring can interact favourably with the pi electrons and partially negatively charged carbons of the other. Metal ions can help to stablize the folded state of proteins as well, as the metal ions are coordinated, usually by lone pair donation from oxygen or nitrogen atoms. Protein stability is showed to have increased in the presence of macromolecular crowding agents. However, the destabilizing influence of crowding in terms of structure and stability of certain macromolecules has also been reported.
We can help you with increasing your protein stability by taking into consideration various factors and rationally designing the protein accordingly. Biochemical and biophysical methods can be used to experimentally assess the stability after proper modifications have been applied.