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Purification of Cyclodextrin Glycosyltransferase by Immunochromatography
Author(s) -
Han Nam Soo,
Tao Bernard Y.
Publication year - 1997
Publication title -
starch ‐ stärke
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.62
H-Index - 82
eISSN - 1521-379X
pISSN - 0038-9056
DOI - 10.1002/star.19970490307
Subject(s) - chromatography , chemistry , affinity chromatography , lysis , cyclodextrin , elution , sepharose , escherichia coli , recombinant dna , cyanogen bromide , enzyme , biochemistry , gene , peptide sequence
Cyclodextrin glycosyltransferase (CGTase) was produced in recombinant Escherichia coli which contains the cgt gene of Bacillus macerans in the pTCGT1 plasmid. Antiserum against CGTase was prepared in rabbits. Anti‐CGTase IgG in Serum was purified using an immobilized Protein A affinity column and an immobilized E. coli cell lysate affinity column. The purified anti‐CGTase IgG was coupled to Sepharose 4B (5.3mg IgG/mL gel), which was activated with cyanogen bromide. Crude CGTase was purified with this immunoadsorbent with CGTase binding capacity of 0.27 mg/mL and eluted with 0.1M glycine buffer, pH 10.6. Approximately 65% of enzyme activity was recovered from the crude CGTase preparation by this method. The purified CGTase was electrophoretically and antigenically homogeneous, appearing as a single band. After 10 cycles, the recovery yield was not reduced significantly. There was no cross reaction between anti‐CGTase IgG and other tested amylolytic enzymes. This immunoaffinity column is, therefore, effective, efficient, and convenient to use, and can be used widely to purify CGTase from various sources.