Isolation of hydrolysing Linamarase enzyme from cassava root peel

Equipment

• Test tubes • Graduated cylinder • Pipets • Balance • Centrifuge (ultracentrifuge)• Filtration devices (including

ultra filters)• 0.45microm membrane pore

Reagents

• Saturated (NH4)2SO4 solution.

• 0.1M acetate buffer(pH 5.5)• 0.1M phosphate buffered

saline PBS

Procedure• Dice peel into small pieces and blend together for 8 mins in 300mL of

acetate buffer to avoid damage of structure• Macro molecules are removed by filtering to remove debris• Ultracentrifuge is used to spin solution at 10,000 rpm for 30mins• More dense molecules migrate away from axis of centrifuge and less

dense move towards axis.

Procedure• The supernatant is carefully decanted out or pipetted out.• The saturated ammonium sulphate is then added continually for salting-out

process. This causes the enzymes to ppt-out• The ppt is dissolved in 50mL PBS and was dialysed(via porous cellulose tubing to

remove remaining salt) using the membrane pore size.• For further purification, the supernatant is spun two more times with a progressive

higher speed and 0.2um membrane(ultrafiltration) is used and components like linamarin permeated out while the pure enzyme which could not pass through is collected as the retentate.

• The enzyme will be stored below 4oC to avoid denaturation.

Isolation of linamarin from cassava

Equipment and materials• Bulk cassava roots• Ultra filter of membrane

size of 0.2um• Blender• Picrate paper• Industrial mill bag• Plastic bucket• evaporator

Reagents• Boiling methanol

Procedure• 100g cassava root is diced and blended together in boiling methanol(99.5%)• Using the mill bags, squeeze out the solution for about an hour in a plastic bucket• Solution settles and clear supernatant is decanted• It is then followed by ultrafiltration using 0.2um set• The isolated linamarin mixture is then evaporated with an evaporator set at 45oC• A dark brown jelly-like solid was recovered.• The linamarin presence is tested with a picrate paper.• The linamarin is impure and needs purification.

Ultrafiltration: membrane technology

• Separation of components from a solution with mixed molecule sizes by flowing the solution under very high pressure over the surface of a membrane.

• The cut-off for ultrafiltration systems is 500-300,000 MW.• The molecular weight of linamarin is 235.4MW.• The cross-flow membrane is preferred to the dead-end flow because it has less tendencies of

feed fouling or concentration polarization.

Purification of linamarin: Adsorption Process

• 80g of isolated and evaporated CCE is dissolved in 250mL distilled water• The solution is then introduced into 80g of ATC in a Pyrex conical flask• A shaking incubator is used on it at 190rpm at 25oC• Solution turns colourless after filtering with 185mm filter paper supported with

vacuum pump.• After each contact time, 4mL filtrate is added to 11mL lis. Solution• Colourless solution obtained is dry frozen with liquid nitrogen to form white

granules

Model: In Vitro Evaluation(expected results)

• HL-60 cells are exposed to only purified linamarin(conc of 300ug/mL) without lis showed a reduction in cell growth by 60%

• With the addition of lis of conc 150ug/mL, cell viability will reduce in excess of 90% is seen.

References1)Iyuke, S. E., Yusuf, U. F., Razil, R., Fakhuru, A. I.,Billa, N and Achike, F. I. (2004).Antitumoral action of linamarin and cassava tissue extracts: Engineering for life. The7th World Congress of Chemical Engineering, Scotland, 140, 2-15.2)Link, N., Aubel, C., Kelm, J. M., Marty, R.R., Greber, D., Djovo, V., Bourhis, J., Weber, Wand Fussenegger, M. (2006). Therapeutic protein transduction of mammalian cells andmice by nucleic acid-free lentiviral nanoparticles, Nuclei Acid Research 43 (2) e16-e16.http://nar.oxfordjournals.org/cgi/content/full/342/e163)Mantell, C. L. (1951). Adsorption. 2nd Ed., McGraw-Hill Book Company, Inc, USA pp 1-321.4)Norzilah, B. A. (2005). Adsorption of 4-nitrophenol onto palm shell based activated carbon.Thesis, Department of Chemical Engineering, University of Malaya, Kuala Lumpor,Malaysia. Pp 11.5) Ralph, T. Y. (2003). Adsorbents: fundamentals and application. John Wiley & Sons, Inc.,Publication, Hoboken, New Jersey. Pp 79-122.