PURIFICATION OF CYCLODEXTRIN GLYCOSYLTRANSFERASE BY IMMUNOCHROMATOGRAPHY

Cyclodextrin glycosyltransferase (CGTase) was produced in recombinant Escherichia coli which contains the cgt gene of Bacillus macerans in t he pTCGT1 plasmid. Antiserum against CGTase was prepared in rabbits. A nti-CGTase IgG in serum was purified using an immobilized Protein A af finity 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 pur ified with this immunoadsorbent with CGTase binding capacity of 0.27 m g/ml and eluted with 0.1M glycine buffer, pH 10.6. Approximately 65% o f enzyme activity was recovered from the crude CGTase preparation by t his method. The purified CGTase was electrophoretically and antigenica lly homogeneous, appearing as a single band. After 10 cycles, the reco very yield was not reduced significantly. There was no cross reaction between anti-CGTase IgG and other tested amylolytic enzymes. This immu noaffinity column is, therefore, effective, efficient, and convenient to use, and can be used widely to purify CGTase from various sources.