Chohan Shahid Mahmood, Rashid Naeem, Sajed Muhammad, Imanaka Tadayuki
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan.
The Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Folia Microbiol (Praha). 2019 May;64(3):313-320. doi: 10.1007/s12223-018-0656-6. Epub 2018 Oct 25.
The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with L-asparaginases. In search of a thermostable L-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100 °C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing D-asparagine with a 20% activity as compared to 100% with L-asparagine. Pcal_0970 did not show any detectable activity when L-glutamine or D-glutamine was used as substrate. Pcal_0970 exhibited a K value of 4.5 ± 0.4 mmol/L and V of 355 ± 13 μmol min mg towards L-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 ± 0.6 kJ mol. To the best of our knowledge, Pcal_0970 is the most thermostable L-asparaginase with a half-life of more than 150 min at 100 °C and this is the first report on characterization of an L-asparaginase from phylum Crenarchaeota.
嗜热栖热放线菌的基因组序列包含两个开放阅读框，即Pcal_0144和Pcal_0970，它们与L-天冬酰胺酶具有同源性。为了寻找一种无谷氨酰胺酶活性的热稳定L-天冬酰胺酶，我们在大肠杆菌中克隆并表达了编码Pcal_0970的基因。重组Pcal_0970以不溶性和无活性的形式产生，经溶解和重折叠后成为具有酶活性的形式。重折叠后的Pcal_0970在100℃及以上温度时表现出最高活性。该酶活性的最适pH为6.5。添加二价金属阳离子或EDTA对活性没有显著影响。该酶能够水解D-天冬酰胺，与L-天冬酰胺相比活性为20%。当使用L-谷氨酰胺或D-谷氨酰胺作为底物时，Pcal_0970没有显示出任何可检测到的活性。Pcal_0970对L-天冬酰胺的K值为4.5±0.4 mmol/L，V为355±13 μmol min mg。根据线性阿伦尼乌斯图确定的活化能为39.9±0.6 kJ mol。据我们所知，Pcal_0970是最耐热的L-天冬酰胺酶，在100℃下半衰期超过150分钟，这是关于泉古菌门L-天冬酰胺酶特性的首次报道。
