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Studies on Luciferase Enzymes for their Application in Assessing Hygiene State of Selected Food Samples.

IR@CSIR-CFTRI

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Relation http://ir.cftri.com/11945/
 
Title Studies on Luciferase Enzymes for their Application in
Assessing Hygiene State of Selected Food Samples.
 
Creator Rajeev, Ranjan
 
Subject 02 Safety
05 Enzymes
 
Description Assurance of public health through the accessibility of safe food and water is an absolute
requirement. Food hygiene is one of the major components of food safety which ensure
prevention of all hazards that make food injurious to health. These hazards include
contamination of food commodities with foodborne pathogens and their toxins, heavy
metals and pesticides. Therefore, rapid and on–site monitoring of food commodities and
water is of prime importance to ensure safe consumption of food and water. Firefly
luciferase enzyme and in vivo bacterial luciferase enzyme based detection methods for
monitoring food hygiene have been considered as the most sensitive and rapid biosensing
tool for hygiene assessment.
In the present study, a novel firefly luciferase enzyme from locally available Asymmetricata
humeralis walker having broad pH optimum and high specific activity (pH optima: 7.4–8.2;
specific activity: 8.15*109 RLU/mg; fold purification: 362.2 and yield: 0.52%) was extracted
and purified to homogeneity. Firefly luciferase enzyme purification was carried out in three
steps viz. ammonium sulphate precipitation (35–85%), DEAE sepharose fast flow column
and blue sepharose column. A novel bioluminescent probe was designed for the detection
of staphylococcal enterotoxin B (SEB) for which ATP was conjugated to SEB using 1–
Ethyl–3–(3–dimethylaminopropyl)carbodiimide (EDC) and N–hydroxysulfosuccinimide
(Sulpho–NHS) method. ATP–SEB conjugate was purified using G–25 sephadex size
exclusion column which was used to develop a dipstick based bioluminescent probe for the
detection of SEB toxin based on competitive Immunoassay.
Further, the purified enzyme was used for rapid enumeration of microbes upto 50–100 CFU
in real food samples such as milk, fruit juice and flavoured milk through ATP assay for
which a novel ATPase inhibitor based bacterial ATP extraction buffer (Ethanol,
benzalkonium chloride and oligomycin A) was formulated. A similar approach for the
evaluation of post mortem time in raw fish as freshness indicator was developed for which
somatic cell ATP extraction buffer (Sodium orthovanadate and EDTA) was optimized. The
somatic cell ATP extraction buffer developed in the present study was found to enhance the
assay sensitivity on an average of 35 fold with respect to control.
In vivo bacterial luciferase activity of bioluminescent bacteria (CFTRI–BIOPHOTO–1;
GenBank: KC617878.1; Photobacterium leiognathi) isolated from marine mussels was
applied as whole cell biosensing element in form of immobilized biophotonic beads for the
prescreening of hazardous materials such as heavy metals [Hg(II), As(V), Cd(II)] and
pesticide (2,4–D) in spiked water samples after submerged cultivation. Using the present
approaches; rapid and sensitive assessment of hygiene can be carried out in food and
water.
 
Contributor Thakur, M. S.
 
Date 2014
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Language en
 
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Identifier http://ir.cftri.com/11945/1/Rajeev%20Ranjan%20Ph.D.%20Thesis%20RF-0316.pdf
Rajeev, Ranjan (2014) Studies on Luciferase Enzymes for their Application in Assessing Hygiene State of Selected Food Samples. PhD thesis, University of Mysore.