Papers by Florentyna Rodrigues
This study focused on optimizing media components enhance the production of cholesterol oxidase b... more This study focused on optimizing media components enhance the production of cholesterol oxidase by Taguchi orthogonal array design. One factor-at-a-time method was used to initially to investigate the effect of fermentation time, inoculum age, inoculum concentration, carbon sources, nitrogen sources and initial pH on biomass growth and enzyme production. The orthogonal array design 1 (OAD1) was used to identify the components required by the bacterium while the orthogonal array design 2 (OAD2) was used for determining the concentration of the identified components for hyper production of cholesterol oxidase. Based on the results obtained a production medium was formulated with the following components, cholesterol (2.0g/L), Tween-80 (4.0g/L), yeast extract (2.0g/L), magnesium sulphate (0.01g/L), zinc sulphate (0.01g/L), mono potassium phosphate (0.1g/L), sodium phosphate dibasic (0.5g/L), calcium chloride (0.01g/L) and ferrous sulphate (1.5g/L). The amount of cholesterol oxidase produced in the optimized medium by the bacterium was three times higher when compared to the amount of enzyme obtained in unoptimized medium.
A high sensitive and stable amperometric biosensor was developed using cholesterol oxidase, chito... more A high sensitive and stable amperometric biosensor was developed using cholesterol oxidase, chitosan and multiwall carbon nano tubes (MWCNT's) in order to determine total cholesterol in serum samples. Cholesterol oxidase was covalently immobilized on MWCNT's; this enzyme was coated over glassy carbon electrode (GCE) and then a layer of chitosan deposited to prevent enzyme leaching. The ChOx/MWCNT/Chitosan was characterized using fourier transformed-infrared (FT-IR), scanning electron microscopy (SEM), atomic force microscopy (AFM), cyclic voltammetry (CV) and amperometric analyses. These studies indicate immobilization of cholesterol oxidase on MWCNTs, an efficient detection method for cholesterol. The optimum performance of the modified electrode was at pH 7.0 at 40°C. The optimized biosensor possessed a minimum detection limit of 0.13µM (S/N=3) with a fast response time (~5s) and a wide linear range 0.16 to 9.69 mM (25 to 500 mg/dL) (N = 10, R2 > 0.99) and 92% stability up to 7 months. The proposed amperometric biosensor is highly sensitive and exhibits no interference with serum metabolites viz. glucose, uric acid, ascorbic acid and billirubin at their physiological concentrations ranges. Thus, the electrochemical biosensor fabricated in the present investigation is promising for cholesterol detection in blood.
The renewed interest in exploring various natural habitat/environments for discovering novel micr... more The renewed interest in exploring various natural habitat/environments for discovering novel microbial sources as stable cholesterol oxidase producers is on the incline due to its broad-range of clinical and industrial applications. A search was conducted to isolate cholesterol oxidase producing bacteria from samples collected from different environments. A total of 98 bacterial isolates capable of degrading cholesterol were obtained from various soil samples collected from in and around Chennai. The bacterial strain producing the highest level of cholesterol oxidase was selected, identified as Enterobacter cloacae by molecular studies and used for all further studies. Purification of cholesterol oxidase was carried out by ammonium sulphate precipitation (80% w/v) followed by Q sepharose and DEAE-cellulose chromatographie techniques. The enzyme was purified to 4.63 fold with a recovery of 93.8 mg of purified protein from 2,889 mg of total protein. The purification yield of the enzyme was 14.86% and its specific activity was 21.8 Umg −1. SDS PAGE and MALDI TOF analyses proved that the enzyme was a monomer with a molecular mass of 56.50461. The enzyme exhibited optimum activity at pH 7.0 and was stable up to 40 º C for an hour. Metal ions such as, Mn + , K + and Zn + inhibited the enzyme activity by 82, 37.66 and 32.8% respectively while most of the other metal ions showed less than 25% inhibition. Triton X-100 and Tween-80 failed to inhibit its activity, however SDS decreased in its activity by 35.2%. Therefore E.cloacae can be exploited for industrial production of stable cholesterol oxidase.
Samples of soil were collected from Varanasi, India. From these samples, 75 different bacterial i... more Samples of soil were collected from Varanasi, India. From these samples, 75 different bacterial isolates were obtained. These isolates were screened for the production of cholesterol oxidase enzyme. Two isolates tested positive for the production of Cholesterol oxidase and the isolate with the higher enzyme production was chosen for this study. The bacterium (isolate GS-14) was identified as Stenotrophomonas maltophila. The bacterium grew on nutrient medium, with cholesterol substrate. Cholesterol oxidase (EC 1.1.3.6), which catalyzes cholesterol into 4-cholesten-3-one, was evidenced from the strain. The conditions for the growth of the bacterium were optimized for extracellular enzyme production, which then reached around 1900 UL −1 within 20 hours of culturing. The enzyme was partially purified from the spent medium of the strain it was run on SDS-PAGE, and characterized. According to this technique, its molecular mass was found to be 20.5 kDa. Cholesterol oxidase showed an optimum activity at pH 7.0.The optimum temperature was found to be 50 o C and it retained more than 70% activity at 50°C after 60 min of heat treatment. The enzyme is pH dependent with maximum activity at around 7.0. Enzyme activity was found to be stable for at least 60 min at pH 7.0 by exhibiting more than 80% activity.
Scientists of agriculture and plant pathology are on the lookout for potential biological control... more Scientists of agriculture and plant pathology are on the lookout for potential biological control agents to control the plant pathogenic organisms in order to avoid soil contamination. Rhizospheric bacteria are excellent agents to control soil-borne plant pathogens. In this study an attempt has been made to evaluate the antagonistic activity of a bacterial strain Bacillus circulans against Curvularia lunata, Alternaria alternata and Cladosporium sp., which are important seed and soil borne pathogens distributed throughout the world. These infections cause grain mold and leaf spot diseases, resulting in significant economic loss to the crops. A soil bacterium Bacillus circulans isolated from the rhizospheric soil of Vigna unguiculata, showed high antagonistic activity against Curvularia lunata, Alternaria alternata and Cladosporium sp. on dual plate assay. Bacillus circulans showed a distinct inhibition zone in the dual plate assay and also produced a clear inhibition zone in chitin amended agar medium containing 0.5% colloidal chitin, indicating that it secretes chitinase. Plant pathogenic fungi cause many dreadful diseases in plants throughout the world. Hence it is crucial to eliminate or to control the plant pathogens so as to increase the quality and yield of the crop. Synthetic chemical based fungicides were extensively used to control fungal diseases however they have deleterious effects on the surrounding environment. The use of synthetic fungicides and other methods were not found to be successful in eradicating these harmful pathogens. Recently, bacterial species have been used in controlling fungal diseases. These micro organisms are capable of lysing chitin, a major component of the fungal cell wall, thus playing an important role in biological control of fungal pathogens. Fungi like Trichoderma, and bacteria like Bacillus, Serratia, and Alteromonas were reported to have chitinolytic activity (Ashwini and Srividhya, 2014). Non-pathogenic soil bacillus species offers several advantages over other organisms as they form endospores and hence can tolerate extreme temperature, pH and osmotic conditions. Bacillus species were found to increase plant growth and cause lysis of fungal mycelia by colonizing the root surface. MATERIALS AND METHODS Sample Collection: The infected plants (Vigna unguiculata) and its own rhizosphere were collected from an agricultural farm in Padalam near Vedanthangal and pathogenic fungi were isolated and grown on potato dextrose agar. Preliminary In vitro Antagonism test: Ten isolates of Bacteria were obtained from Vigna unguiculata rhizosphere and, were evaluated in vitro as antagonists against isolates of Alternaria alternata, Curvularia lunata and Cladosporium sp. Dual cultures were carried out by using one-week-old cultures of Alternaria alternata, and Curvularia sp. on PDA. A 5-mm-diameter disc of the antagonist was positioned opposite a 5-mm-diameter disc of the pathogen on the inoculated agar medium. An approximate distance of 5 cm was maintained between discs. Cultures were grown at 29 ± 2°C, and observed after four days. A sterile disc of agar was placed in the control treatment plate instead of the bacterial isolates. Replicates of three were used for each treatment. At the end of the incubation period, radial growth was measured. The efficiency of Bacteria in suppressing radial growth was calculated as follows: (C _ T)/C · 100 where C is radial growth measurement of the pathogen in the control and T is radial growth of the pathogen in the presence of the test Bacterium.
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Papers by Florentyna Rodrigues