Protein engineering of ascorbate oxidase and its application to clinical diagnostics and food atorage

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The ascorabate oxidase cDNA isolated from Acremonium sp, HI-25 was successfully expressed using Aspergillus nidulans as an intermediate host under the promoter of the Aspergillus oryzae Taka-amylaseA gene. Utilizing this newly developed enzyme production system, we attempted to increase the enzyme productivity, alter the enzyme properties especially pH profile of enzyme catalysis by protein engineering, determine the three dimensional structure and find novel ways to use the enzyme in clinical diagnostics and food storage#l. To increase the enzyme productivity, Hap complex which enhances at least five times the expression level of Taka-amylaseA have been characterized in detail by reconstituting in vitro DNA binding complexes with various combinations of recombinat subunits.#2. Substitution of Valine 193 for Proline in the Substrate Binding Region I(SRBI)was found to shift the pH optimum to neutrality by 1 unit. Then, many amino acid residues in this region was site-specificly mutated, followed by characterization of their pH optima, azide sensitivity and heat stability. The pH optima of V193P and P190I were shifted significantly upward without altering the other enzyme properties.#3. Based on the ASOM structure deduced from computer graphics modelling, Glu192 forms ion pair with Arg309. Replacement of Val193 with Proline could disrupt this ion-pair and result in formation of new stable ion-pairing probably between Glu192 and Arg309. This alteration of the conformation of the specificity pocket influenced the optimum pH of enzyme catalysis.