Optimization and scale up of the adsorption fractionation of cod pyloric caeca deoxyribonuclease using axial and radial flow columns.
The key step in the purification of a deoxyribonuclease (DNase) from extracts of cod (Gadus morhua L.) pyloric caeca, is the selective retention of the enzyme by anion exchange chromatography. The cod DNase purification on Q-Sepharose Fast Flow (Pharmacia) was optimized, using a 60 ml fixed-bed column. In combination with titration curve analysis, we have screened the effect of buffer pHs, feed conductivity and protein loading, on the product recovery and purity. We have developed elution conditions which allow effective separation of the cod DNase from bounded impurities, such as proteinases and nucleic acids. Low levels of these impurities were regarded as essential for the desired product quality. The optimum resolution and maximum purification (ca. 20-fold increase in specific activity) of DNase, was, however, achieved at low protein loading (2.6 mg ml-1 gel), corresponding to less than 4% of the dynamic bed capacity. Scale-up to a 2.5 l pilot scale column (axial flow) and a 0.25 l radial flow column showed that the separation and yield obtained at laboratory scale was retained, and was independent of column geometry and bed height. The implications for a production scale scenario of 100 g of fractionated protein, are also discussed, as well as process hygiene. The optimization described herein adds further knowledge to the treatment of fish waste and the downstream processing of valuable biochemicals from marine raw material.
Department of Biochemistry, University of Bergen, Norway.
K O
KO
Straetkvern
A J
AJ
Raae
K
K
Folkvord
B A
BA
Naess
I M
IM
Aasen
D000327:Adsorption; D000818:Animals; D002852:Chromatography, Ion Exchange; D003851:Deoxyribonucleases / Q000302:isolation & purification*; D005399:Fishes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D012965:Sodium Chloride / Q000494:pharmacology
D012965:Sodium Chloride; D003851:Deoxyribonucleases