Rv2485c, a Putative Lipase of M. tuberculosis: Expression, Purification and Biochemical Characterization
Journal Title: International Journal of TROPICAL DISEASE & Health - Year 2014, Vol 4, Issue 1
Abstract
Lipases have been demonstrated to have a role in virulence in several pathogens. Rv2485c gene product of Mycobacterium tuberculosis has been annotated as putative carboxyl esterase (LipQ) involved in cellular metabolism and respiration. The gene was expressed only in oxidative stress condition in in vitro culture of M. tuberculosis H37Ra as shown by Real Time PCR which suggests its role during dormant stage. Thereby, Rv2485c gene was cloned and expressed in E. coli. The LipQ enzyme was purified as a His-tagged protein from inclusion bodies and refolded with 37% protein yield. The specific activity of purified enzyme was calculated to be 93 U/mg with pNP-palmitate as a preferred substrate. It showed optimum enzyme activity in the range of 40-500C and pH 8.0. The Ser-249, Asp-344 and His-377, predicted as the member of the catalytic triad, were confirmed by site directed mutagenesis. The enzyme was inhibited in the presence of PMSF and DEPC suggesting the presence of Ser and His residues in catalytic site. The apparent Km and Vmax were calculated to be 1.45 mM & 196.08 U/ml respectively. The turnover number (kcat) of the enzyme was calculated to be 6.597 min−1. Based on the results it might be suggested that the LipQ is a lipase, hydrolyzing long chain esters, while the expression of gene only in oxidative stress condition suggested that the enzyme might be playing a role in intracellular survival of microorganism in the human macrophages. The manuscript deals with the detail characterization of oxidative stress inducible lipase and represents a step towards the elucidation of its biological function in vivo.
Authors and Affiliations
Gurdyal Singh, Stuti Arya, Manisha . , Jagdeep Kaur
Phenotypic Characterisation of Escherichia coli Isolates from Fish, Diarrheic and Healthy Children in Zanzibar, Tanzania
Aims: This study was carried out with the objective of investigating E. coli virulence factors, antibiotic sensitivity, presence of extended-spectrum-β-lactamase [ESBL] and serotype H1 O157 in E. coli from fish foods in...
A Retrospective Analysis of Dengue Cases in Suriname: Implications for Treatment and Prevention in a Upper Middle Income Country (UMIC)
Aims: To describe hospitalized dengue cases and characterize the hospitalization length and cost of dengue based on clinical and laboratory classification in a primary hospital in Paramaribo, Suriname. Study Design: A re...
The Predictive Diagnostic and Prognostic Cut-off Values for Interleukin 8 in Patients with Meningitis in Egypt
Objectives: This work was done to detect the best cut-off values for IL-8 to diagnose CNS infections (meningitis and meningio-encephalitis), to differentiate septic from aseptic types, and to predict mortality. Methods:...
Daytime Sleepiness among Healthy Adults in South West Nigeria
Aims: Obstruction of the upper airway during overnight sleep is associated with non-refreshing sleep and daytime sleepiness. This may be an antecedent to accidents, declining quality of life, poor concentration, as well...
Lesion Aspirate Isolation, Culture, Diagnosis and Molecular Identification of Cutaneous Leishmaniasis in District Kohat, Khyber Pakhtunkhwa, Pakistan
Leishmaniasis is a poverty-associated zoonotic tropical disease transmitted by the bite of infected female sand fly. Diagnosis and recognition of clinical manifestation and morphological features of Leishmania parasites...