Abstract

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium ions for thermo-stability, and is a promising alternative to commercially avail-able α-amylases to increase the efficiency of industrial pro-cesses like the liquefaction of starch. We analyzed the amino acid sequence of this α-amylase by sequence alignments and structural modeling, and found that this α-amylase closely resembles the α-amylase from Pyrococcus woesei. The gene of this α-amylase was cloned in Escherichia coli and the re-combinant α-amylase was overexpressed and purified with a combined renaturation-purification procedure. We con-firmed thermostability and exogenous calcium ion indepen-dency of the recombinant α-amylase and further investigated the mechanism of the independency using biochemical ap-proaches. The results suggested that the α-amylase has a high calcium ion binding affinity that traps a calcium ion that would not dissociate at high temperatures, providing a direct expla-nation as to why the addition of calcium ions is not required for thermostability. Understanding of the mechanism offers a strong base on which to further engineer properties of this α-amylase for better potential applications in industrial pro-cesses.

• Keywords: hyperthermophilic α-amylase; Thermococcus sp.; calcium independency