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Protein Separations q Carlson, A.; Nagarajan, R. Release and Recovery of Porcine Pepsin
and Bovine Chymosin from Reverse Micelles: A New
Technique Based on Isopropyl Alcohol Addition. Biotechnology
Progress 8, 85-90 (1992). Related Graduate
Student Thesis Ø
Shaffer, Pamela (M.S. 88)
"Selective Extraction of Proteins Using Micelles" (with A.
Carlson) Ø Kizil, Ramazan (M.S. 98) "Solubilization of Hydrophobic Proteins in Reverse Micelles" |
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Separation of Pepsin +
Chymosin Using AOT Reverse Micelles |
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After complete solubilization into the reverse micelle,
porcine pepsin was not released from AOT in isooctane reverse micelles even
under aqueous-phase conditions which would not ordinarily allow their uptake.
Similarly, bovine chymosin, once
forward-transferred at a pH below its isoelectric
point, was not back-transferred into an aqueous contact phase buffered at a
pH value above its isoelectric point. These results show that there is significant hysteresis
in the forward- and backward-transfer processes and further imply that
kinetics, and not equilibrium, control uptake or release processes for these enzymes. The addition of 10-15 % isopropyl alcohol to the aqueous
phase increases the rate of protein release dramatically and allows for
nearly complete back-transfer of porcine pepsin and 70% back-transfer of
bovine chymosin. IPA addition does not destroy the functional integrity
of the reverse micelle system since forward transfer of bovine chymosin still occurs at pH values below (but not above)
the pl of the protein. The activity of the enzymes is retained in the
presence of the IPA. |
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Chymosin (Renin) pI
= 4.6 Pepsin pI < 2.0 Acid protease used to
catalyze milk clotting in cheese manufacturing (a) HPLC analysis of
original aqueous phase with both chymosin and
pepsin (b) HPLC analysis of
aqueous phase after contact with micellar phase, showing the presence of only
pepsin. (c) HPLC analysis of
back extracted aqueous phase, showing the recovered chymosin |
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