バイオテクノロジーとバイオマテリアル

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Development of Rapid Assay for Ribonucleotide Reduction by Mycobacterium Smegmatis Mc2 155 and their Biochemical Characterisation

Elhachemi Ahmed, Bendaha Mohammed Lamine, Benattouche Zouaoui, Kanoun Khedoudja and Abbouni Bouziane

Mycobacterium smegmatis mc2 155 contains a Ribonucleotide reductases (RNR), which catalyses the irreversible reduction of ribonucleotides to the corresponding 2 �?deoxyribonucleotides required for DNA replication and cell proliferation.

The aim of this work was the development of a rapid assay for ribonucleotide reduction by Mycobacterium smegmatis mc 2 155 and their biochemical characterisation.

For this purpose, the cells of Mycobacterium smegmatis mc 2 155 were permeabilized with two organic solvents toluene and ether for two times (2,5 min) to develop a new assay for ribonucleotide reduction. Due the importance of the growth phase in determining the yield of biomass and ribonucleotide reductase activity of Mycobacterium smegmatis mc 2 155, a correlation between Ribonucleotide reductase activity and growth of Mycobacterium smegmatis mc 2 155 in modified seed medium has been investigated.

For the enrichment of the Ribonucleotide reductase, different purification procedure has been achieved by using fast protein liquid chromatography (FPLC) with superdex G-200 chromatography and Phenyl-Superose HR 5/5 and the enzyme activity was assayed by using (HPLC).

Ribonucleotide reductase activity was detectable in the 40-60% ammonium sulphate fraction. A further purification procedure by gel filtration on the superdex G-200 led to a dissociation of the both subunits. Therefore, a biochemical complementation assay was necessary to identify ribonucleotide reductase activity. The obtained specific activity of the purified protein was 1790 pmol per mg per min with an overall yield of 10%. The purified small subunit of MS2- protein was detected on SDS-PAGE, which was showed a strong band that corresponds to an apparent molecular weight of 38.5 KDa.

The obtained results of ribonuleotide reduction activity with ether permeabilized cells of Mycobacterium smegmatis mc 2 155 presented a comparable enzyme activity for both times, while with toluene permeabilized cells indicated a low enzyme activity. Furthermore, the obtained results of the correlation between ribonucleotide reductase activity and the growth showed that Ribonucleotide redutase is a peak enzyme.

Finally, the permeabilisation of the cells of M. smegmatis mc 2 155 with ether and toluene for short time facilitated us to develop a rapid assay for ribonucleotide reductase activity of others gram positive bacteria.