enzyme immobilization by noncovalent bond on a resin

this presentation give detail on enzyme immobilization on resin and also discuss a research article

1. Introduction and Background Methods Results Conclusion CONTENT

2. Enzyme immobilization has been a captivating research topic since the 1960s BACKGROUND Problems and Limitations Associated with Industrial Enzymes

3. BACKGROUND Methods of Enzyme Immobilization e.g., porous silica, polyacrylamide, agarose, porous glass, etc. caging of enzymes in a network of fibers, through covalent or noncovalent bonds entrapping several biomolecules into different polymeric matrices. interconnected through covalent bonding without carriers via cross-linkers. ionic interaction among the enzymes and the support materials physical adsorption, it possesses a weak binding force ion exchange resins, alumina, activated carbon, and many more

4. Duolite A568 is a highly porous weak base anion resin (particle size 150–600 mm; functional group –N-(R)2) commercially manufactured by DOW chemical company. Enzymes adsorption on this ion-exchange resin surface mainly via electrostatic attractions between the tertiary amine groups of resin and carboxylic acid groups of the enzyme associated with hydrophobic association and Van der Waals' forces. The INTRODUCTION Duolite A568

5. INTRODUCTION crosslinker, poly (ethylene glycol) diglycidyl ether α- glucan phosphorylase phosphoglucomutase

6. Methodology Recombinant enzyme expression Purification of αGP and PGM Enzymatic activities and protein concentration assay Co-immobilization sequences Co-immobilization method Structural characterization of co-immobilized αGP&PGM Optimum temperature and thermal stability Optimum pH Reusability and storage stability of co-immobilized dual enzymes The activities of αGP and PGM were determined to be 106 U and 110 U mixed at the activity ratio of 1:1 dispersed in phosphate buffer (50 mM; 1 mL; pH 7.0) 50 mg carrier ( Duolite A568) was added The mixture was then incubated in a shaker (200 rpm) at 25 °C for 6 h crosslinking agent, PEGDGE solution (0.6%, v/v), was added to the mixture in the shaker and incubated for another 2 h carrier was washed and stored at 4 °C

7. Methodology Co-immobilization methods Adsorption-crosslinking : α GP PGM (0.6%, v/v PEGDGE solution Crosslinking-adsorption: (0.6%, v/v PEGDGE solution Crosslinking-adsorption crosslinking : (0.6%, v/v PEGDGE solution

8. SEM Analysis surface structure of the Duolite A568 resin. magnification of the surface structure. (c) inner structure of the Duolite A568 resin. (d) magnification of the inner structure. (e) surface structure of the co-immobilized αGP&PGM (f) the internal structure of the co-immobilized αGP&PGM.

9. Fourier-transform infrared spectroscopy   ( FTIR) To investigate the chemical constitution and functional groups of co-immobilized dual enzymes.

10. Thermogravimetric analysis (TGA) When PEGDGE concentration was increased from 0.6% to 1.0%, the activity of co-immobilized dual enzymes decreased by nearly 30%.

11. Optimum conditions of the co-immobilization process (a) co-immobilized sequences (b) co-immobilized methods (c) enzyme-activity ratio of PGM/αGP (U/ U) (d) enzyme dosage (e) adsorption temperature (f) adsorption pH

12. Effect of pH and temperature on free and co-immobilized αGP&PGM optimum reaction temperature for free enzymes was 70 °C optimum pH values of free and co-immobilized αGP&PGM were 6.5 and 7.0

13. Thermal stability and Enzyme kinetic of co-immobilized α GP&PGM The Km value of co-immobilized αGP&PGM was 61.95 mM, which was 4.24 times higher than free enzymes (14.61 mM)

14. Reusability and storage stability of co-immobilized dual enzymes The relative activity of co-immobilized αGP&PGM was maintained above 78.5% after eight repeated cycles, Co-immobilized αGP&PGM retained 74.5% activity after two months of storage, whereas the free enzymes were found to be virtually inactivate

15. Simultaneous co-immobilization of αGP&PGM exhibited better catalytic activity compared to other sequencing methods, Relative activity was higher in co-immobilized method of adsorption crosslinking when compared to crosslinking-adsorption and crosslinking-adsorption-crosslinking methods. Higher catalytic activity Enhanced reusability Greater stability Conclusion

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