Cloning,purification and biochemical characterisation of an organic solvent-, detergent-, and thermo-stable amylopullulanase from Thermococcus kodakarensis KOD1 |
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Authors: | Qingtian Guan Xiaohan Guo Ting Han Mengwei Wei Meiling Jin Fan Zeng Lin Liu Zhe Li Yuhan Wang Gang-Won Cheong Shihong Zhang Baolei Jia |
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Affiliation: | 1. College of Plant Sciences, Jilin University, Changchun 130-062, China;2. Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun 130-062, China;3. Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea |
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Abstract: | Thermostable amylopullulanases can catalyse the hydrolysis of both α-1,4 and α-1,6 glucosidic bonds and are of considerable interest in the starch saccharification industry. In this study, the gene Apu-Tk encoding an extracellular amylopullulanase was cloned from an extremely thermophilic anaerobic archaeon Thermococcus kodakarensis KOD1. Apu-Tk encodes an 1100-amino acid protein with a 27-residue signal peptide, which has a predicted mass of 125 kDa after signal peptide cleavage. Sequence alignments showed that Apu-Tk contains the five regions conserved in all GH57 family proteins. Full-length Apu-Tk was expressed in Escherichia coli and purified to homogeneity. The purified enzyme displayed both pullulanase and amylase activity. The optimal temperature for Apu-Tk to hydrolyse pullulan and soluble starch was >100 °C. Apu-Tk was also active at a broad range of pH (4–7), with an optimum pH of ~5.0–5.5. Apu-Tk also retained >30% of its original activity and partially folded globular structure in the presence of 8% SDS or 10% β-mercaptoethanol. The high yield, broad pH range, and stability of Apu-Tk implicate it as a potential enzyme for industrial applications. |
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Keywords: | Amylopullulanase GH57 family Thermophilic archaeon Extremely stable Industrial application |
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