Low-temperature Separation Of Amylase, Purification And Enzymatic Properties

A strain producing cold-adapted amylase was isolated from a washroom of student dormitory in Kunming University of Science and Technology. It was identified as a strain of Arthrobactor based on morphological, physiological characteristics and phylogenetic analysis of 16S rDNA. The new isolated strain was named as Arthrobactor sp. BL5, and the amylase produced by this strain was named as Amy-BL5.Amy-BL5 was an extracellular enzyme. Based on the optimization of various parameters such as starch concentration nutrient components and pH range , the conditions for production of amylase were optimized. The fermentation conditions were established as follow: Na₂HPO₄ 12.8g/L, KH₂PO₄ 3.0g/L, NaCl 0.5g/L, NH₄C1 0.5g/L, soluble starch 10g/L. The optimal temperature for production of cold-adapted amylase was 25℃, the optimal carbon and nitrogen source were soluble starch, optimal fermentation time was 48h. Under this conditions the cold-adapted amylses activity was improved from 5.23U/ml to 27.6U/ml.The amylase was purified to homogeneity from fermentation liquor by ultrafiltration, DEAE Sepharose^TM FF and Sephacryl^TMHR S-100 gel filtration, which has been proved by Native-PAGE activity stain. The specific activity of purified amylase was increased from 405.75U/mg to 8197.1U/mg with purification fold of 20.2 and yield of 11.6%. The molecular mass of Amy-BL5 was determined by SDS-PAGE to be 46.0kD, which is same as Superdex 75 done, so it should be a monomeric protein.Characterization of the Amy-BL5 showed that it had high catalytic activity and stable at temperature of 0℃ to 25℃, the optimal reaction temperature of Amy-BL5 was determined at 25℃. The optimal pH range was 9, and it was stabled from 8 to 10. The amylase activity was activated by K⁺ Na⁺ Ca²⁺ Mg²⁺ Mn²⁺, and was inhibited by Cd²⁺ Co²⁺ Ni²⁺ Pb²⁺ Fe²⁺, SDS EDTA, acetonitrile and DMSO showed partially inhibition on this enzyme, but it was not inhibited by PMSF, it indicated that a srine was not involved in catalytic center of this enzyme.At 25℃, the Km of Amy-BL5 was 1.536mol·L^-1 and Kcat was4.938×10⁵ mol^-1L·S^-1 .The Ea of Amy-BL5 was calculated to be 25.8kJ/mol, indicated that thisenzyme have high affinity to starch substrate. All data showed that Amy-BL5 had a very high catalytic efficiency at low temperature. Based on such characteristics, the enzyme should have a potential value in the industrial applications.