Purification of a amylase—pullulanase bifunctional enzyme by high-performance size-exclusion and hydrophobic-interaction chromatography

Abstract A novel bifunctional enzyme, amylase—pullulanase enzyme (APE), which is produced by Bacillus circulans F-2, was separated and purified in only two steps by high-performance size-exclusion (HPSEC) and high-performance hydrophobic-interaction chromatography (HPHIC) with 50 m M phosphate buffer (pH 7.3) containing 5 m M Co 2+ . About a 90% recovery of the total enzyme activity was achieved, together with a 1821-fold increase in specific activity. APE activity recovered from the column decreased rapidly in the absence of Co 2+ , which acts as an activator and stabilizer. However, most of the activity was restored on the addition of Co 2+ . When a descending salt gradient (1 to 0 M ammonium sulphate in 60 min) and a mobile phase containing Co 2+ were used in HPHIC, the APE characteristics were altered, resulting in earlier elution of the enzyme. The results indicate that the hydrophobic properties of APE can be altered by the addition of Co 2+ , and that the application of HPSEC amd HPHIC with cations such as Co 2+ results in the effective purification of a high-molecular-weight enzyme.