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首页> 外文期刊>Applied Microbiology and Biotechnology >Purification and properties of a thermoactive and thermostable pullulanase from Thermococcus hydrothermalis, a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent
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Purification and properties of a thermoactive and thermostable pullulanase from Thermococcus hydrothermalis, a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent

机译：从深海热液喷口分离出的超嗜热古菌Thermococcus hydrothermalis的热活性和热稳定支链淀粉酶的纯化和性质。

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摘要

The extremely thermophilic archaeon Thermococcus hydrothermalis, isolated from a deep-sea hydrothermal vent in the East Pacific Rise at 21degree CN, produced an extracellular pullulanase. This enzyme was purified 97-fold to homogeneity from cell-free culture supernatant. The purified pullulanase was composed of a single polypeptide chain having an estimated MW of 110 kDa (gel filtration) or 128 kDa (SDS-PAGE). The enzyme showed maximum activity at 95deg C and pH 5.5. The thermostability and the thermoactivity were considerably enhanced in the presence of Ca2+. The enzyme was activated by 2-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and alpha-cyclodextrin were inhibitors. As this enzyme was able to hydrolyse not only the alpha-1,6-glycosidic linkages in pullulan but also the alpha-1,4-glycosidic linkages in amylose and soluble starch, it should be classified as a type II pullulanase or an amylopullulanase. The purified enzyme displayed Michaelis constants of 0.95 mg/ml for pullulan and 3.55 mg/ml for soluble starch without Ca2+, or 0.25 mg/ml for pullulan and 1.45 mg/ml for soluble starch with 1 msmall_capm Ca2+.
机译：极嗜热古生菌Thermococcus hydrothermalis，是从位于21°CN的东太平洋上升区的深海热液喷口中分离出来的，产生了一种胞外支链淀粉酶。从无细胞培养上清液中将该酶纯化97倍至同质。纯化的支链淀粉酶由单链多肽组成，估计分子量为110 kDa（凝胶过滤）或128 kDa（SDS-PAGE）。该酶在95℃和pH 5.5下显示最大活性。在存在Ca 2+的情况下，热稳定性和热活性大大提高。该酶被2-巯基乙醇和二硫苏糖醇激活，而N-溴琥珀酰亚胺和α-环糊精是抑制剂。由于该酶不仅能够水解支链淀粉中的α-1,6-糖苷键，而且还能够水解直链淀粉和可溶性淀粉中的α-1,4-糖苷键，因此应将其分类为II型支链淀粉酶或淀粉支链淀粉酶。纯化的酶显示出米氏常数，对于支链淀粉为0.95 mg / ml，对于不含Ca2 +的可溶性淀粉为3.55 mg / ml，对于支链淀粉为0.25 mg / ml，对于具有1 m small_cap m Ca2 +的可溶性淀粉为1.45 mg / ml。

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