4.5 Article

Purification and characterization of extracellular amylase from the marine yeast Aureobasidium pullulans N13d and its raw potato starch digestion

Journal

ENZYME AND MICROBIAL TECHNOLOGY
Volume 40, Issue 5, Pages 1006-1012

Publisher

ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2006.07.036

Keywords

marine yeasts; glucoamylase; raw starch; purification; characterization

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The extracellular glucoamylase in the supernatant of the cell culture of the marine yeast Aureobasidium pullulans N13d was purified to homogeneity with a 7.3-fold increase in specific activity as compared to the concentrated supernatant by ammonium sulfate fractionation and gel filtration chromatography (Sephadex (TM) G-75). According to the data on native polyacrylamide gel electrophoresis, the molecular weight of the purified enzyme was 98 kDa. However, the data on SDS polyacrylamide gel electrophoresis show that the purified enzyme consisted of two subunits whose molecular weights were 65 and 33 kDa, respectively. The optimal pH and temperature of the purified enzyme were 4.5 and 60 degrees C, respectively. The enzyme was activated by Ca2+, Ba2+, Na+, Cu2+, Mg2+ and Co2+ and stabilized by CaCl2 On the other hand, Hg2+ and Ag+ inhibited the enzyme. The enzyme was inhibited by EDTA, EGTA and SDS, but was not inhibited by iodoacefic acid and PMSF. K-m, and V-max values of the purified enzyme for soluble starch were 5.75 +/- 0.3 mg/ml and 0.25 +/- 0.02 mg/ml/min, respectively. Among raw potato starch, com starch and sweet potato starch tested, all of them were absorbed by the purified enzyme, but only raw potato starch was digested by the purified enzyme. 15.8% of raw potato starch (1.0%, w/v) and 21.1 % of cooked potato starch (1.0%, w/v) were hydrolyzed within 30 min by 0.5 U/mg starch of the purified enzyme and only glucose was detected in the hydrolysate, indicating that the enzyme was glucoamylase with debrariching activity. (c) 2006 Elsevier Inc. All rights reserved.

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