Molecular Cloning, E. coli Expression and Characterization of Thermostable Alanine Aminotransferase from Pyrococcus abyssi

被引:0
作者
Nadeem, Muhammad Shahid [1 ]
Khan, Jalaluddin Azam [1 ]
Anwar, Firoz [1 ]
机构
[1] King Abdulaziz Univ, Fac Sci, Dept Biochem, Jeddah 21589, Saudi Arabia
关键词
Alanine aminotransferase; Molecular docking; Pyrococcus abyssi; NITROGEN USE EFFICIENCY; RECOMBINANT PROTEINS; PURIFICATION; ALT; TRANSCRIPTION; OPTIMIZATION; ALIGNMENT; DOCKING;
D O I
10.17582/journal.pjz/20220217130248
中图分类号
Q95 [动物学];
学科分类号
071002 ;
摘要
Catalysing the reversible interconversion of L-alanine and alpha-ketoglutarate to pyruvate and L-glutamate in the presence of pyridoxal 5 phosphate, alanine aminotransferase (ALT) is an enzyme which operates at the cross roads of amino acid and carbohydrate metabolism. The enzyme has been reported from a wide range of organisms including animals, plants, fungi and microbes. The enzyme has a clinical applications in the diagnosis of many diseases. In the present study we have produced a recombinant of ALT from Pyrococcus abyssi in BL21 (DE3) strain of E. coli. The recombinant enzyme was purified by anion exchange chromatography, it displayed a 45kDa band on SDS-PAGE, with 58.1% final recovery, 15.3 fold purification and 139 U/mg specific activity. Maximum enzyme activity was found at pH 8 and above 90C, its KM and Vmax values were found 25 mu M L-alanine and 149 U/min/mg respectively. In silico studies have shown that the enzyme was found in a monomer structure. Molecular docking studies with potential molecules involved in the reaction catalysed have been conducted and binding energy values were calculated for each molecule including L-alanine, pyridoxal 5 phosphate, pyruvate, alpha ketoglutarate and L-glutamate. Present study provides the first report of ALT from Pyrococcus abyssi and suggests active site residues of enzyme from archaeal origin.
引用
收藏
页码:871 / 880
页数:10
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