The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium

Huaixu Cheng; Zhidan Luo; Mingsheng Lu; Song Gao; Shujun Wang

doi:10.1007/s12275-017-6416-5

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The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium: Huaixu Cheng ^1,2,3, Zhidan Luo ^1,3,4, Mingsheng Lu ^1,2,3, Song Gao ^1,2,3, Shujun Wang ^1,2,3; Journal of Microbiology 2017;55(5):379-387.
DOI: https://doi.org/10.1007/s12275-017-6416-5
Published online: March 1, 2017

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. China¹Jiangsu 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

Corresponding author: Song Gao , Tel: +86-518-85586628,
Shujun Wang , Tel: +86-518-85586628,

Received: 23 August 2016 • Revised: 19 December 2016 • Accepted: 11 January 2017

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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

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The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium

J. Microbiol. 2017;55(5):379-387. Published online March 1, 2017

DOI: https://doi.org/10.1007/s12275-017-6416-5

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