title: The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium.
abstract: The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium ions for thermostability, and is a promising alternative to commercially available α-amylases to increase the efficiency of industrial processes 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 recombinant α-amylase was overexpressed and purified with a combined renaturation-purification procedure. We confirmed thermostability and exogenous calcium ion independency of the recombinant α-amylase and further investigated the mechanism of the independency using biochemical approaches. 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 explanation 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 processes.
authors.affiliation: Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, Lianyungang, 222005, P. R. China.|Jiangsu Marine Resources Development Research Institute, Lianyungang, 222005, P. R. China.|Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang, 222005, P. R. China.
authors.forename: Huaixu
authors.initials: H
authors.lastname: Cheng
authors.affiliation: Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, Lianyungang, 222005, P. R. China.|Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang, 222005, P. R. China.|Lianyungang City Academy of Agricultural Sciences, Lianyungang, 222001, P. R. China.
authors.forename: Zhidan
authors.initials: Z
authors.lastname: Luo
authors.affiliation: Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, Lianyungang, 222005, P. R. China.|Jiangsu Marine Resources Development Research Institute, Lianyungang, 222005, P. R. China.|Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang, 222005, P. R. China.
authors.forename: Mingsheng
authors.initials: M
authors.lastname: Lu
authors.affiliation: Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, Lianyungang, 222005, P. R. China. gaosong@hhit.edu.cn.|Jiangsu Marine Resources Development Research Institute, Lianyungang, 222005, P. R. China. gaosong@hhit.edu.cn.|Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang, 222005, P. R. China. gaosong@hhit.edu.cn.
authors.forename: Song
authors.initials: S
authors.lastname: Gao
authors.affiliation: Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, Lianyungang, 222005, P. R. China. shujunwang86@163.com.|Jiangsu Marine Resources Development Research Institute, Lianyungang, 222005, P. R. China. shujunwang86@163.com.|Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang, 222005, P. R. China. shujunwang86@163.com.
authors.forename: Shujun
authors.initials: S
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology*; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D000516:alpha-Amylases; D002118:Calcium
keywords: Thermococcus sp.; calcium independency; hyperthermophilic α-amylase