HYDROPHOBIC INTERACTION CHROMATOGRAPHY ON OCTYL SEPHAROSE—AN APPROACH FOR ONE-STEP PLATFORM PURIFICATION OF CYCLODEXTRIN GLUCANOTRANSFERASES

Abstract

Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans ATCC 21783 was concentrated by ultrafiltration and subsequently purified by hydrophobic interaction chromatography on Octyl Sepharose 4 fast flow. The matrix was able to bind selectively to the enzyme at a very low ammonium sulfate concentration of 0.67 M and enzyme desorption was performed by decreasing gradient of the salt. The overall recovery was 80% with 689-fold purity. CGTases derived from four soil isolates and Toruzyme, the commercial preparation of CGTase, also bound to Octyl Sepharose under similar conditions at 0.67 M and eluted at 0.55–0.5 M of ammonium sulfate. Octyl Sepharose chromatography can thus be used as a platform approach for purification of CGTases from various bacterial sources. Long stretches of sequence predominated by hydrophobic amino acids are reportedly present in the starch binding domains of CGTases. Starch binding experiments indicated the binding of the enzymes to the octyl matrix through these domains.

Keywords:

• alkalophilic Bacillus
• cyclodextrin glucanotransferase
• hydrophobic interaction chromatography
• Octyl Sepharose
• purification
• starch binding domain

Notes

^a Mean of three determinations.

^b Hydrophobic interaction chromatography.

^c Specific activity in U/mg.

Note. SBD, starch binding domain.

Note. Experimental conditions are given in text. One unit of the ultrafiltrate was incubated with 0.5 g of the starch. For the incubation period of 10 min, intermittent shaking was performed every 2 min. Supernatent was assayed for dextrinising activity to determine the unadsorbed activity. Amount of β-CD formed by the bound enzyme was estimated by phenolphthalein complexation method.

^a B, Difference between the applied unit and the unbound units = 1 – U.

^b Milligrams of β-CD formed/hr by bound enzyme at 50°C.

Note. Ultrafiltrate containing 0.9/1.2 units was incubated with 0.2 mL of the resin in phosphate buffer containing 0.67 M ammonium sulfate in the presence or absence of starch. The enzyme bound to the resin was desorbed and was analyzed for bound activity. Experimental conditions are given in text.

^a Dextrinizing activity in the desorbate, which reflects the bound activity.

^b Milligrams of β-CD formed/hr by desorbed enzyme at 50°C.