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Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates
Author(s) -
Chang Y. K.,
McCreath G. E.,
Chaset H. A.
Publication year - 1995
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260480408
Subject(s) - elution , expanded bed adsorption , adsorption , chromatography , yeast , chemistry , yield (engineering) , dehydrogenase , enzyme , biochemistry , organic chemistry , materials science , metallurgy
The use of an expanded bed of STREAMLINE Red H‐7B for the purification of the intracellular glycolytic enzyme glucose 6‐phosphate dehydrogenase (G6PDH) directly from untreated preparations of disrupted yeast cells has been investigated. Small‐scale experiments, carried out in packed beds, have shown that the optimal pH for adsorption is 6.0 and have enabled optimization of elution conditions using a series of eluents. The dynamic capacity of the adsorbent for G6PDH was determined in a small expanded bed to be 28 units/mL. These results were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 expanded bed column. G6PDH was purified directly from an unclarified yeast homogenate in 99% yield with an average purification factor in the eluted fraction of 103. Cleaning‐in‐place (CIP) procedures using 0.5 M NaOH and 4 M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity of alteration of bed expansion characteristics after many cycles of operation. © 1995 John Wiley & Sons, Inc.