Rv2485c, a Putative Lipase of M. tuberculosis: Expression, Purification and Biochemical Characterization
Gurdyal Singh, Stuti Arya, Manisha ., Jagdeep Kaur
International Journal of TROPICAL DISEASE & Health · pp. 1–17 · Published 9 Oct 2013
10.9734/IJTDH/2014/5808Abstract
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.
References (42)
- 1 Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method [DOI]
- 2 Molecular Cloning: A Laboratory Manual
- 3 Basic local alignment search tool [DOI]
- 4 CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice [DOI]
- 5 Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence [DOI]
- 6 Protein structure prediction on the Web: a case study using the Phyre server [DOI]
- 7 Immunology of Tuberculosis [DOI]
- 8 Cold Spring Harbor Laboratory [DOI]
- 9 Mycobacterium tuberculosis Uses Host Triacylglycerol to Accumulate Lipid Droplets and Acquires a Dormancy-Like Phenotype in Lipid-Loaded Macrophages [DOI]
- 10 A Novel In Vitro Multiple-Stress Dormancy Model for Mycobacterium tuberculosis Generates a Lipid-Loaded, Drug-Tolerant, Dormant Pathogen [DOI]
- 11 A secreted lipase of Fusarium graminearum is a virulence factor required for infection of cereals [DOI]
- 12 Mechanisms of bacterial pathogenicity [DOI]
- 13 Mycobacterium tuberculosis inhibits IFN-gamma transcriptional responses without inhibiting activation of STAT1.
- 14 BIOCHEMICAL DIFFERENTIATION OF MYCOBACTERIUM TUBERCULOSIS GROWN IN VIVO AND IN VITRO [DOI]
- 15 A Novel Lipase Belonging to the Hormone-sensitive Lipase Family Induced under Starvation to Utilize Stored Triacylglycerol in Mycobacterium tuberculosis [DOI]
- 16 Acid Resistance inMycobacterium tuberculosis [DOI]
- 17 Human Granuloma In Vitro Model, for TB Dormancy and Resuscitation [DOI]
- 18 Differential Gene Expression in Response to Exposure to Antimycobacterial Agents and Other Stress Conditions among SevenMycobacterium tuberculosis whiB-Like Genes [DOI]
- 19 Functional Role of the PE Domain and Immunogenicity of the Mycobacterium tuberculosis Triacylglycerol Hydrolase LipY [DOI]
- 20 Triacylglycerol Utilization Is Required for Regrowth of In Vitro Hypoxic Nonreplicating Mycobacterium bovis Bacillus Calmette-Guerin [DOI]
- 21 Lipid hydrolizing enzymes in virulence:Mycobacterium tuberculosisas a model system [DOI]
- 22 Lipase activity of thermophilic bacteria from icelandic hot springs [DOI]
- 23 Silencing of Oxidative Stress Response in Mycobacterium tuberculosis : Expression Patterns of ahpC in Virulent and Avirulent Strains and Effect of ahpC Inactivation [DOI]
- 24 Occurrence of a lipase in spores ofAlternaria brassicicolawith a crucial role in the infection of cauliflower leaves [DOI]
- 25 Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties [DOI]
- 26 Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids [DOI]
- 27 Lipolytic enzymes in Mycobacterium tuberculosis [DOI]
- 28 Two cutinase‐like proteins secreted by Mycobacterium tuberculosis show very different lipolytic activities reflecting their physiological function [DOI]
- 29 Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis [DOI]
- 30 Mycobacterial lipolytic enzymes: A gold mine for tuberculosis research [DOI]
- 31 Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis [DOI]
- 32 LipC (Rv0220) Is an Immunogenic Cell Surface Esterase of Mycobacterium tuberculosis [DOI]
- 33 Temperature stability of proteins essential for the intracellular survival of Mycobacterium tuberculosis [DOI]
- 34 Evidence for the cytotoxic effects of Mycobacterium tuberculosis phospholipase C towards macrophages [DOI]
- 35 Interference ofStaphylococcus aureus lipase with human granulocyte function [DOI]
- 36 Production of lipase by clinical isolates of Pseudomonas cepacia [DOI]
- 37 Expression and characterization of the carboxyl esterase Rv3487c from Mycobacterium tuberculosis [DOI]
- 38 Thermostable Hexameric Form of Eis (Rv2416c) Protein of M. tuberculosis Plays an Important Role for Enhanced Intracellular Survival within Macrophages [DOI]
- 39 The lipF promoter of Mycobacterium tuberculosis is upregulated specifically by acidic pH but not by other stress conditions [DOI]
- 40 Induction of an extracellular esterase from Candida albicans and some of its properties [DOI]
- 41 Case of Low Fever: Delirium: Incomplete Dementia: Convulsions: Death: Tuberculosis [DOI]
- 42 [Hydrolysis of fatty acids esters by Mycobacterium phlei].
Cited by 14
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Gurkamaljit Kaur, Varinder Saini, Bandana Kumari · International Journal of Biological Macromolecules · 2017
Ruchi Rastogi, Gurkamaljit Kaur, Pratibha Maan · Journal of Medical Microbiology · 2019
Stuti Arya, Parul Singh, Jashandeep Kaur · Molecular Biology Reports · 2022
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