High-Temperature Crystallization Method for the GH57 Family Hyperthermophilic Amylopullulanase from Aquifex aeolicus

Abstract

(Hyper)thermophilic enzymes derived from (hyper)thermophilic microorganisms have significant implications for both basic research and industrial applications. Investigating the thermal stability and catalytic mechanisms of thermophilic enzymes from a structural perspective is essential for the rational modification of these enzymes. Currently, X-ray crystallography remains one of the key techniques for determining the structures of biomolecules; however, protein crystallization is a multiparameter-regulated and often unpredictable process. Here, we present a method for the high-temperature crystallization of the GH57 family hyperthermophilic amylopullulanase from Aquifex aeolicus (AaApu), conducted at 338 K. In comparison to traditional crystallization methods performed at 277 or approximately 291 K, the crystals grown at high temperatures are larger, with dimensions increasing from 400 × 10 μm² to 200 × 150 μm². Additionally, the time required for crystal growth is significantly reduced from 5 days to 2 h, and there is a notable improvement in resolution, enhancing from 2.5 to 1.7 Å. Furthermore, high-temperature crystallization facilitates the binding of larger molecular weight substrates to the protein molecules and reveals the catalytic mechanisms of enzymes. Overall, high-temperature crystallization preserves the original structure of thermophilic proteins, suggesting that it could be a promising method for thermophilic proteins.