[Purification and properties of recombinant extremely thermostable and acid-stable amylase].

ABSTRACT

Extremely thermostable and acid-stable a-amylase produced by Pichia pastoris GS115/pPIC9K-Amy-228 was purified to electrophoretic homogeneity by the steps of ultrafiltration and PAGE. Purification of about 11.7 fold was achieved with an overall yield of 29.8%. Its molecular weight was estimated to be about 55kD by SDS-PAGE. The isoelectric point was 5.0 (room temperature). Michaelis constant of the enzyme for soluble starch was 1.12g/L. The carbohydrate content was 15.4% by the phenol-sulfuric acid method. The optimum temperature and pH of the enzyme activity were 95 degrees C and 4.5 respectively. The enzyme activity was stable under room temperature in the pH rang of 4.0 - 7.0 for 48 hours. About 60% of the initial enzyme activity was measured after 1h of incubation at 110 degrees C. The activity was strongly inhibited by Fe2+, Cr2+ and Cu2+, While Ca2+ had no effect on it. DTT and EDTA had no effect on the activity.