Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris

The medaka fish alpha-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant alpha-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K-M and V-max values were determined: chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (alpha/beta)(8) barrel fold, as do other known alpha-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) alpha-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein. (C) 2012 Elsevier B.V. All rights reserved.