Heterologous gene expression and characterization of TK2246, a highly active and thermostable plant type l-asparaginase from Thermococcus kodakarensis
Heterologous gene expression and characterization of TK2246, a highly active and thermostable plant type l-asparaginase from Thermococcus kodakarensis
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DOI：
10.1016/j.ijbiomac.2020.01.012
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发表时间：
2020-03-15
影响因子：
8.2
通讯作者：
Rashid, Naeem
中科院分区：
文献类型：
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作者：
Chohan, Shahid Mahmood;Sajed, Muhammad;Rashid, Naeem
柯达热球菌的基因组序列包含两个可能的基因TK1656和TK2246，注释为L-天冬酰胺酶。此前曾报道过TK1656。目前的报道集中在植物型L天冬酰胺酶TK2246，它由918个核苷酸组成，对应一个由306个氨基酸组成的多肽。克隆了该基因，并在大肠杆菌中表达，纯化后的基因产物用于鉴定重组酶的性质。TK2246对L-天冬酰胺的最适作用温度为85℃，最适pH为7.0，比活性为767mU·moL·min~(-1)·mg~(-1)。该酶对D-天冬酰胺的活性为10%，而对L-天冬酰胺的活性为100%。未观察到对L或D-谷氨酰胺的活性。TK2246的表观K-m和V-max分别为3.1 mM和833mU·mol·min~(-1)·mg~(-1)。由Arrhenius图确定的反应活化能为28.3kJ·mol~(-1)。据我们所知，这是首次从Eurya chaeota门的热球菌类中鉴定出一种植物型L天冬酰胺酶。(C)2020爱思唯尔B.V.保留所有权利。
The genome sequence of the hyperthermophilic archaeon Thermococcus kodakarensis contains two putative genes, TK1656 and TK2246, annotated as L-asparaginases. TK1656 has been reported previously. The current report is focused on TK2246, a plant-type L-asparaginase, which consists of 918 nucleotides corresponding to a polypeptide of 306 amino acids. The gene was cloned, expressed in Escherichia coli and the purified gene product was used to determine the properties of the recombinant enzyme. TK2246 was optimally active at 85 degrees C and pH 7.0 with a specific activity of 767 mu mol min(-1) mg(-1) towards L-asparagine. The enzyme exhibited a 10% activity towards d-asparagine as compared to 100% against l-asparagine. No detectable activity was observed towards L- or D-glutamine. Half-life of the enzyme was nearly 18 h at 85 degrees C. TK2246 exhibited apparent K-m and V-max values of 3.1 mM and 833 mu mol min(-1) mg(-1), respectively. Activation energy of the reaction, determined from the Arrhenius plot, was 28.3 kJ mol(-1). To the best of our knowledge, this is the first characterization of a plant-type L-asparaginase from class Thermococci of phylum Euryarchaeota. (C) 2020 Elsevier B.V. All rights reserved.