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Isolation of symbiotic bacteria and bioactive proteins from

Isolation of symbiotic bacteria and bioactive proteins from

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Published by: Nidhi Vaidya on Feb 11, 2011
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02/11/2011

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Isolation of symbiotic bacteria and bioactive proteins from the marine sponge, Callyspongia diffusa

NIDHI VAIDYA G.E.09016

Escherichia coli. coli. Vibrio cholera. SDSSDS-PAGE was used to know the molecular weight to the proteins extracted       . Vibrio parahaemolyticus cholera.Basic idea of the paper  Symbiotic association aeruginosa. Pseudomonas aeruginosa. Bioactive proteins in methanol and in aqueous extract antibacterial property hemolytic activity on chicken erythrocytes Protein estimation was carried out.

Mumbai coast Handpicking of the Callyspongia diffusa during low tide Preservation at low temperature Boiled with concentrated HNO3 to extrude the spicules and confirm the identity based on the characters proposed by Thomas    .Methods and materials  Study area ± khardhanda beach near Juhu.

Nutrient agar. Vibrio agar.Isolation of the associated bacteria Fresh tissue of sponge cut by sterile blade Homogenized using pestle and mortar Serial dilution pour in different sterile media plates [Mac Conkey¶s agar. Eosin methyl blue agar] Bacterial associates were identified using Bergey¶s manual .

 Methanolic extraction Extraction of crude toxin  Aqueous extraction (bakus and green) sponge dried in air(2days) 10g tissue soaked in 200ml of methanol(5hrs) Removal of solvent by squeezing the sponge filter through Whatman filter no.1 solvent evaporation at low pressure using Buchi Rotavopor at 60°C 60° Storage of extract in refrigerator for further use Squeezing the sand free sponge specimens in triple distilled water Solution obtained is filtered and dialyzed using sigma dialysis membrane 500 against D-glucose Dto remove excess water supernatant obtained is lyophilized using the Labcono Freeze Dry System Stored at 4°C in refrigerator for further 4° use .

001 ml of each sponge extract Then placed in centre of the inoculated Petri dish Bacterial colonies were allowed to grow overnight at 37°C 37° Inhibition zone around the disc was observed .Antimicrobial activity Petri dish with sterile nutrient agar was inoculated with the isolated bacteria Sponge extract was sterilized by passing each through a 0.22µm Millipore GV filter Round paper discs were dipped into 0.

Partial purification of crude extract  Partial purification was carried out using DEAE cellulose anion exchange chromatography according to the method of Stempion .

5ml of dilute folin¶s phenol reagent & mix well Incubate for 30 minutes Take absorbance at 650nm spectrophotometrically Protein was estimated using standard graph .1 to 1 mg/ml 5ml of alkaline copper reagent was added Mix well and allow it to stand for 10 minutes Add 0.Protein Estimation (by Lowry & Lopez method) Standard used was Bovine albumin serum at concentration ranging from 0.

Hemolytic activity Performed in 96 well µv¶ bottom microtitre plates Selection of different rows for chicken blood 2-fold serial dilution of crude toxin in 100ml of normal saline and repetition of above steps up to the last well 100µl RBC was added to all wells Appropriate controls were included 1% RBC suspension & 100µl normal saline negative control Gentle shaking of plates And allowed to stand for 2 hrs at room temperature Recorded the results (uniform red color suspension positive hemolysis Button formation at bottom lack of hemolysis) Reciprocal of highest dilution of crude toxin showing pattern was taken as 1 hemolytic unit (HU) .

75mm thickness) overlaid with 5% stacking gel.  .SDSSDS-PAGE  One dimension Sodium dodecyl sulphate polyacrylamide gel electrophoresis was carried out Proteins were electrophoresed on 12% separating gel (0.

. version-13.0 followed by versionDuncan¶s multiple range test (DMRT).Statistical analysis  Statistical analysis was carried out using one way analysis of variance (ANOVA) by SPSS software package.

greenish 2grey on VA E.Result  Morphological and physiological characteristics of the bacteria isolated from the sponge species are given : Bacteria P. 1-2mm diameter. MAC. coli V. aeruginosa Colony characters Growth on NAM light-grey lightcolonies of 1. cholera . 1swarm across plate Colonies are round smooth 2-3mm. parahaemolytic us V.5mm diameter Metallic sheen colonies on EMB agar Grey colonies on NAM.

The crude methanolic extract induced hemolysis on chicken erythrocytes and its hemolytic titre was 14 and also its hemolytic activity was estimated at 8.46 HU/mg of protein whereas the hemolytic titre of aqueous extract was 10 and its activity was estimated at 6. The 2 extract were tested at different concentration(5. cholera.09g. cholera.4 g of crude extract.99 HU/mg of protein    .62 mg/ml in methanolic extract and 1. The protein content from the sponge was found to be 1. whereas aqueous extract yielded 5. Methanolic extract (500g) of sponge yielded 5.43 mg/ml in aqueous extract.10 and 15 mg/ml) against the isolated bacteria and none of the extract inhibited the test bacteria except the aqueous extract did inhibit V.

3 well defined bands of 19.0. 39. 66.2 kDa in both extract  .5.4 to 116 kDa molecular weight. SDSSDS-PAGE 6 bands in aqueous extract and 8 bands in methanolic extract ranging from 14.

Mebs haemagglutination and hemolytic activity of aqueous extract (48 tropical sponge species). halistanol sulphate and sokotrasterol sulphate (halichondriidae sponge). Suberea preatense Protein content corresponding data on protein content of sponge is not available in literature for comparison Hemolytic activity of both extract are in accordance with earlier studies [Stempein halitoxin (Haliclona). derivatives viz.4g (water) of crude extract compare Richet 5.Discussion  500g sponge yielded 5.   .8g of crude extract from 490g fresh weight of marine sponge. Fusetanie sterol (Haliclona).09g(methanol) and 5..

   .Conclusion  Hemolytic activity indicates cytotoxicity of the extract worthwhile for studies on their antitumour/anti-neopastic antitumour/antiactivities detection of anticancer compounds Antibacterial agents produced by sponge enhancing efficiency with sponge retains bacterial food. The 66.2 kDa band obtained in SDS-PAGE analysis SDSin present study might be stress induced protein and this type of stress induced protein has been reported in marine catcatfish. Marine sponge have anti microbial activity against marine bacteria but no activity against bacteria has been observed thus confirming the isolated bacteria are symbiotic to marine sponge studied.

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