文摘
The medaka fish ¦Á-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 ¦Á-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 (¦Á/¦Â)8 barrel fold, as do other known ¦Á-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) ¦Á-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.