| Title | The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium |
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| Author | Huaixu Cheng1,2,3, Zhidan Luo1,3,4, Mingsheng Lu1,2,3, Song Gao1,2,3*, and Shujun Wang1,2,3* |
| Address | 1Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, Lianyungang 222005, P. R. China, 2Jiangsu Marine Resources Development Research Institute, Lianyungang 222005, P. R. China, 3Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang 222005, P. R. China, 4Lianyungang City Academy of Agricultural Sciences, Lianyungang 222001, P. R. China |
| Bibliography | Journal of Microbiology, 55(5),379-387, 2017, |
| DOI | 10.1007/s12275-017-6416-5 |
| Key Words | hyperthermophilic α-amylase, Thermococcus sp., calcium independency |
| 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. |