Abstract:
Acyl CoA dehydrogenase is the enzyme carrying out the first and rate limiting step in fatty acid metabolism to produce enoyl CoA from acyl-CoA by a,ß-dehydrogenation of acyl-CoA esters and introducing trans double-bond between C2 (a) and C3 (ß) of the acyl-CoA thioester. The presence of acyl CoA dehydrogenase in archaea is a clue for the presence of active fatty acid metabolic pathway in archaea, which otherwise are thought to be absent in archaea. In this study we cloned and characterized Acyl CoA dehydrogenase to identify its role in archaea. Pcal_1308 was 1224 nucleotides in length and encodes a protein of 407 amino acids with a calculated molecular mass of 45 kDa. Pcal_1308 was cloned in pTZ57/RT and 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. For enzymatic activity of recombinant protein, decrease on in A600 was followed in a reaction mixture containing PMS DCIP, FAD, acyl-CoA and 2 µg of purified protein. The effect of temperature on the activity of Pcal_1308 was observed at different temperatures (30ºC-80ºC) by keeping the pH constant. Optimum pH was found by assaying at different pH. The cloning and heterologous expression of Pcal_1308 in BL21CodonPlus and futher analysis of recombinant protein on SDS-PAGE showed a recombinant Pcal_1308 protein of about 45 kDa. By heat treatment of total cell lysate having recombinant protein, Pcal_1308 was found to be thermostable. Acyl CoA dehydrogenase from P. calidifontis was found to be active at high temperature showing maximum activity at optimum temperature 50 ºC. The substrate specificity profile of Pcal_1308 showed that its activity increases with carbon chains up to six then the activity decreases with the longer chained substrates and belongs to medium chain acyl CoA dehydrogenases. The structure of protein was determined by homology modelling by submitting sequence on SwissProt, Expasy using acyl CoA dehydrogenase from homo-sapien (2VIG) as template. The superimposition of the predicted structure of Pcal_1308 with 2VIG (homo-sapien) shows the protein have almost same structure as the characterized protein.Conclusion: The results suggested that Pyrobaculum calidifontis possesses a gene Pcal_1308 that produces an active acyl CoA dehydrogenase, which gives a clue for presence of active fatty acid metabolism in this Archaeon.
Page(s):
91-91
DOI:
DOI not available
Published:
Journal: Abstract Book on Global Science Technology and Management Conference, Volume: 0, Issue: 0, Year: 2023
Keywords:
overexpression
,
Cloning
,
Pyrobaculum Calidifontis
,
Acyl CoA dehydrogenase