Stabilization Of Proteins

The intrinsic instability of some proteins can be problematic for some food processing applications, eg, the use of a less-stable enzyme would require more time and larger amounts of enzyme to achieve the same processing goal as compared to a more stable enzyme. Following the introduction of site-directed mutagenesis in 1982 (85), research focused on the improvement of stability of proteins by site-specific mutations rather than random mutations. This approach was based on the hypothesis that the stability of proteins was governed by (1) bonds and interactions, (2) conformational factors, and (3) protection by modifications (86). To test this hypothesis, new disulfide bonds were introduced to stabilize various proteins, eg, T4 lysozyme

(87,88), subtilisin (89-91), dihydrofolate reductase (92), and X repressor (93).

In a series of experiments, Matthews et al. stabilized T4 lysozyme using different strategies, eg, a-helix dipoles, entropy, conformation strain, and hydrophobic interactions (94,95). An 8°C increase in Tm (melting temperature) using a combination of strategic mutations was achieved (96-101). By changing the hydrophobic interaction between the subunits of L-lactate dehydrogenease, a 10°C increase in Tm was achieved (102). As an alternative type of hydrophobic interaction manipulation, site-specific glyco-sylation of hen egg white lysozyme resulted in a dramatic increase of thermostability (103). Suzuki et al. reported that the introduction of proline could stabilize protein by stabilizing the secondary structure (104). T4 lysozyme was stabilized by introducing a proline residue into an a-helix (105). Nakamura et al. also showed that Bacillus neutral protease was stabilized by introducting proline into a core a-helix (106).

Despite many successful reports on the stabilization of protein by site-directed mutagenesis, there is still no general theory to predict the site to be mutated. Gilis and Rooman used the solvent accessibility of the residues to determine the mutation site (107,108). The prediction of stabilization was based on a database containing the results of the mutation studies of various proteins.

Fersht's group, using barnase, conducted a series of mutation studies on their effects on stability and concluded the effects were varied even in a certain kind of the interactions, depending on the context (109,110). Furthermore, research based on the this hypothesis required that the three-dimensional structures be known in order to determine the mutation site.

Future research could provide a general theory on protein stabilization. Serrano et al. suggested one such possible theory. Systematic multiple mutation could stabilize proteins without the knowledge of the three-dimensional structures but would be dependent on two proteins with high sequence similarity (111). The reader is referred to the article by Jiminez-Flores and Bleck regarding recent advances in food protein biotechnology (112).

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