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Title: Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis

Abstract

Phosphoglucose isomerase (PGI) is a well-characterized ubiquitous enzyme involved in the glycolytic pathway. It catalyzes the reversible isomerization of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate and is present in all living cells. However, there is interspecies variation at the level of the primary structure which sometimes produces heterogeneity at the structural and functional levels. In order to evaluate and characterize the mycobacterial PGI, the gene encoding the PGI from Mycobacterium tuberculosis H37Rv was cloned in pET-22b(+) vector and expressed in Escherichia coli. The target DNA was PCR amplified from the bacterial artificial chromosome using specific primers and cloned under the control of T7 promoter. Upon induction with IPTG, the recombinant PGI (rPGI) expressed partly as soluble protein and partly as inclusion bodies. The rPGI from the soluble fraction was purified to near homogeneity by ion-exchange chromatography. Mass spectrum analysis of the purified rPGI revealed its mass to be 61.45 kDa. The purified rPGI was enzymatically active and the specific activity was 600 U/mg protein. The K {sub m} of rPGI was determined to be 0.318 mM for fructose-6-phosphate and the K {sub i} was 0.8 mM for 6-phosphogluconate. The rPGI exhibited optimal activity at 37 deg C and pH 9.0, and did notmore » require mono- or divalent cations for its activity.« less

Authors:
 [1];  [1];  [1];  [1]
  1. Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067 (India)
Publication Date:
OSTI Identifier:
20713445
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 337; Journal Issue: 2; Other Information: DOI: 10.1016/j.bbrc.2005.09.092; PII: S0006-291X(05)02114-5; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CATIONS; DNA; ENZYMES; ESCHERICHIA COLI; FRUCTOSE; GLYCOLYSIS; ION EXCHANGE CHROMATOGRAPHY; ISOMERIZATION; MASS SPECTRA; MYCOBACTERIUM TUBERCULOSIS; PH VALUE; PHOSPHATES; POLYMERASE CHAIN REACTION

Citation Formats

Mathur, Divya, Ahsan, Zaid, Tiwari, Madhulika, and Garg, Lalit C. Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis. United States: N. p., 2005. Web. doi:10.1016/j.bbrc.2005.09.092.
Mathur, Divya, Ahsan, Zaid, Tiwari, Madhulika, & Garg, Lalit C. Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis. United States. https://doi.org/10.1016/j.bbrc.2005.09.092
Mathur, Divya, Ahsan, Zaid, Tiwari, Madhulika, and Garg, Lalit C. Fri . "Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis". United States. https://doi.org/10.1016/j.bbrc.2005.09.092.
@article{osti_20713445,
title = {Biochemical characterization of recombinant phosphoglucose isomerase of Mycobacterium tuberculosis},
author = {Mathur, Divya and Ahsan, Zaid and Tiwari, Madhulika and Garg, Lalit C},
abstractNote = {Phosphoglucose isomerase (PGI) is a well-characterized ubiquitous enzyme involved in the glycolytic pathway. It catalyzes the reversible isomerization of D-glucopyranose-6-phosphate and D-fructofuranose-6-phosphate and is present in all living cells. However, there is interspecies variation at the level of the primary structure which sometimes produces heterogeneity at the structural and functional levels. In order to evaluate and characterize the mycobacterial PGI, the gene encoding the PGI from Mycobacterium tuberculosis H37Rv was cloned in pET-22b(+) vector and expressed in Escherichia coli. The target DNA was PCR amplified from the bacterial artificial chromosome using specific primers and cloned under the control of T7 promoter. Upon induction with IPTG, the recombinant PGI (rPGI) expressed partly as soluble protein and partly as inclusion bodies. The rPGI from the soluble fraction was purified to near homogeneity by ion-exchange chromatography. Mass spectrum analysis of the purified rPGI revealed its mass to be 61.45 kDa. The purified rPGI was enzymatically active and the specific activity was 600 U/mg protein. The K {sub m} of rPGI was determined to be 0.318 mM for fructose-6-phosphate and the K {sub i} was 0.8 mM for 6-phosphogluconate. The rPGI exhibited optimal activity at 37 deg C and pH 9.0, and did not require mono- or divalent cations for its activity.},
doi = {10.1016/j.bbrc.2005.09.092},
url = {https://www.osti.gov/biblio/20713445}, journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 2,
volume = 337,
place = {United States},
year = {2005},
month = {11}
}