Abstract
Purification of cyclodextrin glucanotransferase (EC 2.4.1.19) from the culture liquid of Bacillus megaterium was carried out by the use of three purification procedures: (a) reverse osmosis and organic solvents precipitation, (b) ultrafiltration and gel-filtration chromatography, and (c) ultrafiltration, adsorption on starch, ion exchange chromatography and gel-filtration chromatography. Best precipitation was achieved by i-propanol – 85.92% of initial enzyme activity was preserved with purification of 1.47-fold. PS-20000 appeared to be the most suitable membrane for ultrafiltration. After 8.3-fold concentration by volume, the enzyme was purified 2.46-fold with a yield of 95.30%. As a result of the two-step purification procedure (ultrafiltration with membrane PS-20000 and gel-filtration chromatography) the enzyme was purified 36.2-fold with 95.45% of enzyme activity [measured in micrograms of cyclodextrins formed in 1 ml in 1 min under the assay conditions(U)] preserved. A highly purified enzyme preparation (purification of 74.56-fold and specific activity of 60.39 U/mg protein) was obtained by the third purification procedure. The purified enzyme preparation showed a single protein band on SDS-PAGE, 10% polyacrylamide gel.
Published Version
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