Kinetic Stabilization of Bacillus licheniformis α-Amylase through Introduction of Hydrophobic Residues at the Surface

Abstract

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis ␣-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13°C higher than the wild-type. Crystal structure analysis at 1.7 Å resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a ␤-sheet by introducing a residue with high ␤-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.Some general rules for increasing the stability of proteins have been derived from a large number of comparative structural and mutagenesis studies (1, 2). Among the most generally recognized strategies for protein thermostabilization are: increasing the hydrophobic packing in the interior, decreasing surface hydrophobicity, extending networks of salt-bridges and hydrogen bonds, engineering disulfide bonds or metal binding sites, shortening or strengthening solvent-exposed loops and termini, increasing the extent of secondary structure formation, and replacing residues responsible for irreversible chemical alterations of the protein structure. Yet, these general rules may not always be applied successfully and examples of engineered mutations resulting in effects opposite to the ones expected are legions. Such studies have also failed to reveal outstanding features associated with the adaptation of proteins to a given temperature range, i.e. psychrophilicity, mesophilicity, thermophilicity, and hyperthermophilicity. While most natural proteins seem to achieve their respective stability by accumulating a large number of weakly stabilizing interactions that result in a large net effect, some have acquired specialized structural features that cannot easily be transferred in a general way into other proteins (3, 4). Elucidating the origin of thermal stability for a given protein and finding ...

Thermostability of Irreversible Unfolding α-Amylases Analyzed by Unfolding Kinetics

“…Based on this, mutant studies often revealed enhanced stabilities that are determined kinetically rather than thermodynamically. For example, recently a BLA mutant was studied that unfolds 32 times slower than the wild type (44). …”

Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

“…The Hansch p-parameter (logP) for NH 3 + groups is log P ¼ -2.12; and for NHCOCH 3 groups log P ¼ -1.21 (Hansch and Steward 1964;Hansch and Coats 1970). Although it is a general rule of protein engineering that increasing the surface hydrophobicity (e.g., by introducing hydrophobic residues via site-directed mutagenesis) decreases the thermostability of the native state (Matthews 1993;Lee and Vasmatzis 1997), BLA represents a well-known exception to this rule Machius et al 2003). Over nine different hydrophobic variants of BLA have been shown to be more thermostable than the wild-type (WT) protein.…”

One-Step Production of Immobilized α-Amylase in Recombinant Escherichia coli

“…While they are widely distributed in plants, animals, and microorganisms, the secreted ␣-amylase from Bacillus licheniformis (BLA) is the one most extensively used in the starch industry (5), due mainly to its thermostability (7, 13). However, BLA has been subjected to protein engineering approaches in order to enhance its technically relevant enzyme properties such as greater temperature stability and a wider pH activity range (16,24).Industrial enzymes are frequently immobilized onto solid supports in order to increase resistance to fluctuations in conditions such as pH and temperature (18) and to facilitate repeated usage. Although BLA has previously been successfully immobilized (25,27,28), it has been suggested that access to large starch molecules through pores in typical supports can be a limitation (27).…”