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 UNDER THE GUIDANCE OF

DR.ANTONY ADLAGATTA DR.N.W.FADNAVIS

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Immobilization of enzymes on to polymers

Immobilization of G FAT enzyme

Purification of GP 11 HSD enzyme andimmobilization.

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L-glutamine + F6-phosphate L-glutamate + D-glucosamine 6-phosphate

Glutamine-fructose-6-phosphate transferase isan enzyme that catalyzes the chemical reaction

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500mg of IB-150 in 200mgof BSA/20 ml Phosphatebuffer(0.05M,pH 7.5)and shake it overnight.

stir it with 10mM DTT(0.0154gm DTT in 10 ml sodium

acetate buffer of 0.1M,pH 4.5 and 0.1M NaCl) for 2hours

wash the DTT treated polymer for 10 to 15 times withsodium acetate buffer (0.1 M ,pH 7.8) and then confirmthe absence of DTT by testing it with DTNB reagent.

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Take 5 mg/ml DSC in DMC and add it to polymer followedby shaking for 2 hours at 4 C

then take 250 mg gfat enzyme in 2 ml of sodiumcarbonate buffer(0.1 M, pH 9.1)allow it to stay for 2hours.

Gfat Assay

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Materials:

Buffer solution: 0.1M KH2PO4,0.05M KCL1mM EDTA

L-Glutamine stock solution-100mM

Fructose 6 phosphate-10mM

Borate buffer-0.8M

NaHCO3(10%)

Ehrlich Reagent(2.5 gm of para dimethyl amino

benzaldehyde +21.9ml glacial acetic acid+3.2ml of conc Hcl)

5% acetic anhydride

enzyme solution 250mg/2 ml

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Buffer(µl) F6P(µl) L-glutamine(µl)

930 30 40

PROCEDURE

Firstly mix the solution as given above.then incubate it for 3 hoursfollowed by adding 0.1 ml of cold NAHCO3 and 0.1 ml ofAcetic

anhydride.then mix it for 5 min at RT and then for 5 min at 80 C.then add 0.5 ml of borate buffer and keep it at 80 C for 7min.

Then finally add Ehrlich reagent.Pink colour shows that thereaction is positive and enzyme is active.

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RECYCLING OF THEIMMOBILIZED ENZYME WASDONE FOR 7 TIMES

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Biobarriers Bioremediation

Waste water treatment

Study and control of chronic bacterialinfection.

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 It is the only technology available totreat ground water through biobarrierin future.

It is a clean up technology

It is a cost effective and reliableTechnique

Bacteria converts ammonium basedfertilizers to nitrogen gas

Making the water pure.

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The larger, roughly sphericalcells that appear green to brownare algae while the smaller darkcells are associated bacteria.

Both types of cells produce apolymeric extracellular slimelayer which encloses the cells.

This complex aggregate of cellsand polysaccharide is the biofilm

community.

These biofilms can have a highaffinity for pollutants such asmetal ions and toxic organiccompounds.

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Thiobacillus ferrooxidans is also capable of oxidizing reduced sulfur species to sulfate asillustrated in the following reactions:

H2S + 2O2 2- + 2H+

So + H2O + 1.5 O2 2- + 2H+

S2O3 2- + H2O + 2O2 2- + 2H+

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•

Single - celled organisms exhibit two modes of behavior:(i) Free-floating(ii) Attached state

• The change in behavior is triggered by many factors including

quoram sensing and other mechanism ,that vary betweenspecies.

• Generally microbes attaches to the surfaces and develops biofilms.

• Firstly colonists adhere to the surfaces initially through weak vanderwaals force .

• They attach themselves using cell adhesion molecules such aspili

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How do biofilms form?