Author(s):
1. Uswa Al:
Department of Biochemistry, IBBB, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
2. Asma Yaqoob:
Department of Biochemistry, IBBB, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
3. Raza Ashraf:
Department of Biochemistry, IBBB, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
Abstract:
Enoyl-CoA hydratases/ Crotonases are the enzyme, playing most important role in fatty acid metabolism to produce an acetyl-CoA in each cycle of beta-oxidation. Enoyl-CoA Hydratase/ Crotonase is also an essential enzyme component of CoA-dependent butanol biosynthesis in microbial cells. The presence of Enoyl-CoA Hydratase/ Crotonase in the Archaea lead to a clue for the presence of active fatty acid metabolic pathways in Archaea, which otherwise are thought to be absent in Archaea. In this study we cloned and characterized Enoyl CoA Hydratase/ Crotonase to identify its role in Archaea. Pcal_1306 encodes a protein of 263 amino acids with a calculated molecular mass of 29 kDa. Pcal_1306 was cloned in pTZ57/RT and then in pET-21a (+) expression vector. E. coli BL21 CodonPlus was used to express the gene. The protein was partially purified by heat treatment and anion exchange chromatography. The enzymatic activity of the recombinant Enoyl-CoA hydratase was assayed for the hydration of the crotonyl-CoA. 50 mM of the Tris-HCl (pH-8.0) and observing fall in OD263 . Hydratase assay was carried out at different temperatures and pH range to find out optimum conditions. Kinetic parameters of the Pcal_1306 were calculated at 80 °C and at pH 7.5, by observing the initial velocities with different concentrations of crotonyl-CoA i.e 0.025-20mM). End product analysis was also carried out through HPLC. The cloning and heterologous expression of Pcal_1306 in E.coli BL21 CodonPlus and further analysis of recombinant protein on SDS-PAGE showed a recombinant Pcal_1306 protein of about 29 kDa. By heat treatment of total cell lysate containing recombinant protein Pcal_1306 was found to be thermostable. Enoyl-CoA Hydratasefrom P. calidifontis was found to be active at high temperature showing maximum activity at optimum temperature 80 ºC. It found to be active on broad range of pH (6.0-9.0) with maximum activity at pH 7.5 for hydration of crotonyl-CoA. Kinetic parameters measurement of the recombinant Enoyl-CoA Hydrataseshowed Vmax and Km which were 130µmol.min-1.mg-1 and 10.18µM, respectively. The structure of protein was determined by homology modelling by submitting sequence on SwissProt, Expasy using crotonase from M. tuberculosis as template. The superimposition of the predicted structure of Pcal_1306 with 3PZK (M. tuberculosis) shows the protein have almost same structure as the characterized protein. The results suggested that Pyrobaculum calidifontis possesses a gene Pcal_1306 that produces an active Enoyl-CoA Hydratase, which gives a clue for presence of active fatty acid metabolism in this Archaeon.
Page(s):
93-93
DOI:
DOI not available
Published:
Journal: Abstract Book on Global Science Technology and Management Conference, Volume: 0, Issue: 0, Year: 2023
Keywords:
Pyrobaculum calidifontis
,
overexpression
,
Cloning
,
Enoyl CoA Hydratase
References:
References are not available for this document.
Citations
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