IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF SIALIDASE (NEURAMINIDASE) GENE AND ENZYME FROM Listeria monocytogenes
IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF SIALIDASE (NEURAMINIDASE) GENE AND ENZYME FROM Listeria monocytogenes
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Date
2014-08
Authors
OKERE, Christiana
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Abstract
A putative Trans-sialidase gene was identified in Listeria monocytogenes, a human and animal
pathogen and its product was characterized. The identification was carried out using Basic Local
Alignment Search Tool (BLAST), Polymerase Chain Reaction (PCR), sequencing and gene
analysis/predictions. The enzyme was partially purified from the supernatant of the cultured
organism by ammonium sulphate precipitation and gel filtration, the enzyme eluted as a single
peak with an activity of 1.6 μM /min. The enzyme was active between pH 4.0 and 4.5, a 45.8%
loss of enzyme activity was found at pH 10.0. and an optimum temperature of 30°C, activity
gradually decreased with increasing temperature which was reduced to 26% at 70°C and was
almost completely lost at 80°C. Effect of cations on the enzyme activity showed that Hg2+ and
Al3+ at 3mM concentration are potent inhibitors while Mn2+, Zn2+ and Ca2+ have no effect on the
enzyme activity at same concentration compared to the control. The release of the enzyme from
the Listeria monocytogenes was time dependent, increasing to 44 × 10-3μM within 60 minutes of
incubation. The Trans-sialidase gene was amplified and ligated into pJET1.2 blunt cloning vector
to obtain a recombinant DNA which was used to transform E.coli DH5α. The transformed DH5α
were selected, the recombinant DNA re-extracted was digested with HindIII and subcloned into
pET-28b (+). The sequence analysis revealed a 342bp encoding a 114 translated amino acids
protein with a calculated molecular mass of 12.132 kDa and isoelectric point of 8.4. A BLASTP
search versus a non-redundant protein sequence database revealed that this protein shows the
highest sequence identity to TS2 gene of Trypanosome congolense (100%). 59.6% for T. vivax,
T. brucei, and T. evansi, while T.cruzi showed 50% and Sialidase gene of T. rangeli 55.1%. All
together, these results suggest that Listeria monocytogenes Trans sialidase might be an important
virulence factor and may be involved in the pathogenesis of this organism
Description
A THESIS SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES,
AHMADU BELLO UNIVERSITY ZARIA NIGERIA IN PARTIAL FULFILMENT FOR
THE AWARD OF MASTER OF SCIENCE DEGREE IN BIOCHEMISTRY
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF SCIENCE
AHMADU BELLO UNIVERSITY, ZARIA
AUGUST, 2014
Keywords
IDENTIFICATION,, MOLECULAR,, SIALIDASE,, NEURAMINIDASE,, ENZYME,, Listeria,, monocytogenes