Abstract
Currently, there are no versatile and established methods for improving stability of proteins. In an entirely different approach from conventional techniques such as mutagenesis, we attempted to enhance enzyme stability of α-amylase from Aspergillus oryzae using a heavy-atom derivatization technique. We evaluated changes in stability using differential scanning calorimetry (DSC). Candidate heavy atoms were identified using the Heavy-Atom Database System HATODAS, a Web-based tool designed to assist in heavy-atom derivatization of proteins for X-ray crystallography. The denaturation temperature of α-amylase derivatized with gadolinium (Gd) or samarium (Sm) ions increased by 6.2 or 5.7°C, respectively, compared to that of the native protein (60.6°C). The binding of six Gd ions was confirmed by X-ray crystallography of the enzyme at 1.5 Å resolution. DSC and dynamic light-scattering data revealed a correlation between stability and the aggregation state upon addition of Gd ions. These results show that HATODAS search is an effective tool for selecting heavy atoms for stabilization of this protein.
Highlights
Enzymes are currently used in many different industrial products and processes [1]
There are no established methods that can be used to predict the effect of mutations on thermal stability, because even single amino-acid substitutions affect various stabilization factors depending on the surrounding environment, substituted residues, and structural changes due to the mutations [4,5,6]
Because heavy atoms sometimes act as inhibitors, we first investigated whether derivatized proteins retained their enzymatic activity
Summary
Enzymes are currently used in many different industrial products and processes [1]. To enhance thermal stability of highly useful enzymes for industrial applications, protein-engineering techniques such as site-directed mutagenesis, random mutagenesis, recombination, and directed-evolution techniques have been successfully employed for various proteins [2,3]. We believe that HATODAS search could contribute to the efficient selection of candidate heavy atoms for use in protein stabilization using the derivatization technique. To elucidate thermal behavior of a derivatized protein, we used differential scanning calorimetry (DSC) to investigate the stability of Aspergillus oryzae a-amylase (Ao aamylase) containing heavy atoms.
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