UNDER THE GUIDANCE OF

DR.ANTONY ADLAGATTA

DR.N.W.FADNAVIS

Immobilization of enzymes on to polymers Immobilization of G FAT enzyme Purification of GP 11 immobilization. HSD enzyme and

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

L-glutamine + F6-phosphate

L-glutamate + D-glucosamine 6phosphate

500mg of IB-150 in 200mgof BSA/20 ml Phosphate buffer(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 2 hours

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

Take 5 mg/ml DSC in DMC and add it to polymer followed by shaking for 2 hours at 4 C then take 250 mg gfat enzyme in 2 ml of sodium carbonate buffer(0.1 M, pH 9.1)allow it to stay for 2 hours. Gfat Assay

Materials: Buffer solution:

0.1M KH2PO4,0.05M KCL 1mM 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+3.2 ml of conc Hcl) 5% acetic anhydride enzyme solution 250mg/2 ml

PROCEDURE
Buffer(l)
930

F6P(l)
30

L-glutamine(l)
40

Firstly mix the solution as given above. then incubate it for 3 hours followed by adding 0.1 ml of cold NAHCO3 and 0.1 ml of Acetic 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 7 min. Then finally add Ehrlich reagent. Pink colour shows that the reaction is positive and enzyme is active.

RECYCLING OF THE IMMOBILIZED ENZYME WAS DONE FOR 7 TIMES

Biobarriers Bioremediation Waste water treatment Study and control of chronic bacterial infection.

It is the only technology available to treat ground water through biobarrier in future. It is a clean up technology It is a cost effective and reliable Technique Bacteria converts ammonium based fertilizers to nitrogen gas Making the water pure.

The larger, roughly spherical cells that appear green to brown are algae while the smaller dark cells are associated bacteria. Both types of cells produce a polymeric extracellular slime layer which encloses the cells. This complex aggregate of cells and polysaccharide is the biofilm community. These biofilms can have a high affinity for pollutants such as metal ions and toxic organic compounds.

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

H2S + 2O2 SO4 2- + 2H+

So + H2O + 1.5 O2 SO4 2- + 2H+ S2O3 2- + H2O + 2O2 2SO4 2- + 2H+

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 between species. 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 as pili

How do biofilms form?