The extracellular cellulase produced, using 5 liters medium was subjected to ammonium sulfate fractionation.
Thus over all two samples Sample A and Sample B were obtained which were subjected to ammonium sulfate fractionation and processed further.
As a result of ammonium sulfate fractionation of Sample A and Sample B, two fractions were obtained from each at 60% saturation and 80% saturation respectively.
Table: 1 Progress of ammonium sulfate fractionation of cellulose of T.
This formed an adequate basis of bulking the growth samples up to 8th day as sample A and those from to 9th to 21st as sample B, which were subjected to ammonium sulfate fractionation and it's binding with DEAE cellulose.
Using the same procedure, solid ammonium sulfate was added to the supernatant to obtain, sequentially, 20% increments of ammonium sulfate fractionation.
Ammonium sulfate fractionation of the extract indicated the presence of toxicity toward P.
herreni are most likely proteins or peptides since they were thermo-labile and were precipitated by ammonium sulfate fractionation, and had molecular weights greater than 3.
After repeated
ammonium sulfate fractionation, the sample was loaded on to the hydrophobic column (Resource PHE), and it eluted as a single peak (Fig.
r-hCK-MB was purified from cell lysate by three chromatographic methods (hydrophobic, ion-exchange, and gel-filtration) after ammonium sulfate fractionation.
r-hCK-MB was purified from crude extract by ammonium sulfate fractionation, followed by hydrophobic, anion-exchange, and gel-filtration chromatography (Table 2).
Purification step Volume, mL Total protein, (a) mg Crude extract (c) 1410 35 532 Ammonium sulfate fractionation Supernatant at 40% saturation 1360 20 550 Precipitate at 80% saturation (d) 338 2075 Phenyl-Sepharose CL-413 207 684 Q-Sepharose 98 218 Sepharcryl S-200 24 48 Purification step Total CK activity, (b) U Yield, % Crude extract (c) 579 200 Ammonium sulfate fractionation 100 Supernatant at 40% saturation 466 793 Precipitate at 80% saturation (d) 46 958 81 Phenyl-Sepharose CL-413 42 290 81 Q-Sepharose 19 221 73 Sepharcryl S-200 6894 (e) 33 12 Purification step Specific Purification, activity, U/mg fold Crude extract (c) Ammonium sulfate fractionation 16 1.
