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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 , Zhidan Luo , Mingsheng Lu , Song Gao , Shujun Wang
- 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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- 9 Crossref
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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.
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Citations
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- Enhanced Thermostability of Geobacillus stearothermophilus α-Amylase by Rational Design of Disulfide Bond and Application in Corn Starch Liquefaction and Bread Quality Improvement
Mengyu Zhu, Wenxin Zhai, Runfei Song, Lin Lin, Wei Wei, Dongzhi Wei
Journal of Agricultural and Food Chemistry.2023; 71(48): 18928. CrossRef - Structural and functional adaptation in extremophilic microbial α-amylases
Aziz Ahmad, Rahamtullah, Rajesh Mishra
Biophysical Reviews.2022; 14(2): 499. CrossRef - Biochemical characterization of an α-fucosidase PsaFuc from the GH29 family
Xiaohua Liu, Xin Geng, Weizhi Liu, Qianqian Lyu
Process Biochemistry.2022; 122: 258. CrossRef - Amylases from thermophilic bacteria: structure and function relationship
Bhavtosh A. Kikani, Satya P. Singh
Critical Reviews in Biotechnology.2022; 42(3): 325. CrossRef - Biochemical and synergistic properties of a novel alpha‐amylase from Chinese nong‐flavor Daqu
Lanchai Chen, Zhuolin Yi, Yang Fang, Yanling Jin, Kaize He, Yao Xiao, Dong Zhao, Huibo Luo, Hui He, Qun Sun, Hai Zhao
Microbial Cell Factories.2021;[Epub] CrossRef - Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications
Suraj Mital, Graham Christie, Duygu Dikicioglu
Microbial Cell Factories.2021;[Epub] CrossRef - A new GH13 subfamily represented by the α-amylase from the halophilic archaeon Haloarcula hispanica
Štefan Janeček, Barbora Zámocká
Extremophiles.2020; 24(2): 207. CrossRef - Contribution of domain B to the catalytic properties of a Flavobacteriaceae α-amylase
Huijia Yin, Lina Zhang, Zhou Yang, Shannan Li, Xinyu Nie, Yan Wang, Chunyu Yang
Process Biochemistry.2018; 70: 104. CrossRef - Functional and cooperative stabilization of a two-metal (Ca, Zn) center in α-amylase derived from Flavobacteriaceae species
Huijia Yin, Zhou Yang, Xinyu Nie, Shannan Li, Xuyang Sun, Chao Gao, Zenghang Wang, Guangming Zhou, Ping Xu, Chunyu Yang
Scientific Reports.2017;[Epub] CrossRef
- Enhanced Thermostability of Geobacillus stearothermophilus α-Amylase by Rational Design of Disulfide Bond and Application in Corn Starch Liquefaction and Bread Quality Improvement
Research Support, Non-U.S. Gov't
- Identification of an Extracellular Thermostable Glycosyl Hydrolase Family 13 α-Amylase from Thermotoga neapolitana
- Kyoung-Hwa Choi , Sungmin Hwang , Hee-Seob Lee , Jaeho Cha
- J. Microbiol. 2011;49(4):628-634. Published online September 2, 2011
- DOI: https://doi.org/10.1007/s12275-011-0432-7
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- 7 Scopus
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Abstract
- We cloned the gene for an extracellular α-amylase, AmyE, from the hyperthermophilic bacterium Thermotoga neapolitana and expressed it in Escherichia coli. The molecular mass of the enzyme was 92 kDa as a monomer. Maximum activity was observed at pH 6.5 and temperature 75°C and the enzyme was highly thermostable. AmyE hydrolyzed the typical substrates for α-amylase, including soluble starch, amylopectin, and maltooligosaccharides. The hydrolytic pattern of AmyE was similar to that of a typical α-amylase; however, unlike most of the calcium (Ca2+)-dependent α-amylases, the activity of AmyE was unaffected by Ca2+. The specific activities of AmyE towards various substrates indicated that the enzyme preferred maltooligosaccharides which have more than four glucose residues. AmyE could not hydrolyze maltose and maltotriose. When maltoheptaose was incubated with AmyE at the various time courses, the products consisting of maltose through maltopentaose was evenly formed indicating that the enzyme acts in an endo-fashion. The specific activity of AmyE (7.4 U/mg at 75°C, pH 6.5, with starch as the substrate) was extremely lower than that of other extracellular α-amylases, which indicates that AmyE may cooperate with other highly active extracellular α-amylases for the breakdown of the starch or α-glucans into maltose and maltotriose before transport into the cell in the members of Thermotoga sp.
- Purification and Characterization of α-amylase from Aspergillus sp. JP-1
- Park, Hyung Nam , Yoo, Jin Cheol , Yang, Young Ki
- J. Microbiol. 1995;33(1):80-84.
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Abstract
- The α-amylase was purified from Aspergillus sp. JP-1 and some enzyme characteristics were studied. The enzyme waw approzimately purified 80-fold and an overall yield was 16.5% from the culture medium by ammonium sulfate fractionation, Sephadex G-150 gel permeation chromatography, and DEAE-Sephadex A-50 ion exchange column chromatography in order. The molecular weight of the purified α-amylase has been estimated to be 56 KDa on SDS-polyacrulamide gel electrophoresis and Sephadex G-150 chromatography. The purigied enzyme functions optimally at pH5.5 and 40℃, respectively. The Km value for soluble starch was 2.5 mg/ml. The enzymatic activity increased in the presence of Ca^2+, Co^2+, EDTA, Mg^2+, Mn^2+ and Zn^2+ and was inhibited by adding Cu^2+, Fe^2+, and Ni^2+.
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