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

The medaka fish α-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 α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and KM and Vmax 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 (α/β)8 barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-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.

► High-throughput construction of secretory protein expression system in P. pastoris
► Alpha-amylase of medaka fish was secretory expressed using the developed method.
► Purified recombinant protein exhibited starch hydrolysis activity.
► Structure of purified recombinant protein was determined by X-ray crystallography.
► Enzymatic property and overall structure are very similar to those of mammal.