Expression,Purification And Enzymatic Analysis Of The Protein Rv1400c From Mycobucterium Tuberculosis

Tuberculosis?TB?,caused by Mycobacterium tuberculosis infection,has a long historyand caused a huge financial loss and mental stress to human beings.TB is hardly cured for many reasons.The most important thing thatM.tuberculosis has a high capacity to evade the host immune system and establish a chronic,latent infection by formed granulomathat a major histopathological hallmark of TB.Most of bacteria in this structureexisted with persistent state and survived for decades till the host's immune system is weakened and active tuberculosis.The persistent bacteria preferentially metabolize lipids rather than carbohydrates metabolize reputed to be an energy source during latency.Considerable research has focused on the lipids present in mycobacteria.In the M.tuberculosis life cycle,the importance of lipids has been established.The cytosol has been found to be an important lipid storage location,forming organites known as lipid inclusion bodies.These organites are essentially composed of triacylglycerols?TAG?.TAG are convenient carbon and energy storage compounds,because they show low rates of oxidation and a high calorific value.These TAG are hydrolysed reflecting the presence of an intracellular lipase.In vivo,the storage of these lipids could be important for the bacteria and enables them to adapt to the environment by preparing them to enter in the dormant phase.TAG that could provide free fatty acid?FA?under starvation conditionsuseful for the constitution of membrane lipids,but also for the synthesis of wax ester under iron limitation.Lipolytic enzymes are distributed throughout the living organisms which form two primary divisions of the phylogenetic tree,namely the bacteria and a second divisionbranching into both the eukarya,including animals,plants and fungi,and the archaea,with the former archaebacteria.The comparative sequence analysis of the M.tuberculosisgenome has revealed that it contains 250 enzymes involved in lipid metabolism,while only 50 in Escherichia coli.Lipid metabolism can be assumed tobe one of the major pathways in mycobacteria and needs tobe studied in detail.However,due to the absence of in vivo study,there is no information whether this enzyme plays a role in the viability or in the infection processes?persistence or reactivation?.Rv1400c?LipI?,which annotated as a putative lipase of Mycobacterium tuberculosis,was cloned,expressed and purified in Escherichia coli as fusion protein.Sequence analysis showed that LipI is a member of subfamily the Hormone Sensitive Lipase?HSL?.The enzyme showed the preference for short carbon chain substrates with optimal activity at 37? / pH 8.0 and stability between pH 6.0 to 9.0 by enzyme assay.The specific activity of enzyme was calculated to 35.7 U/mg with pNP-butyrate as a preferred substrate.The activity was sharply inhibited by SDS,CTAB and Zn²⁺respectively in the use of detergents and metal cation.The catalytic triad of Rv1400 c consists of residues Ser¹⁶⁵ and His²⁹¹ demonstrated by amino acid sequence alignment and site-directed mutagenesis.It will not only improve our understanding of subfamily HSL lipases,but also provides an ideal biocatalyst for the enrichment of polyunsaturated fatty acids.