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

以米曲霉Taka-amylaseA基因为启动子，以中性曲霉为中间宿主成功表达了从Acremonium sp . HI-25中分离得到的抗坏血酸氧化酶cDNA。利用这个新开发的酶生产系统，我们试图通过蛋白质工程来提高酶的产量，改变酶的性质，特别是酶催化的pH谱，确定酶的三维结构，并找到将酶用于临床诊断和食品储存的新方法# 1。为了提高酶的生产效率，Hap复合物可以提高Taka-amylaseA至少5倍的表达水平，通过重组各种重组亚基组合的体外DNA结合复合物对其进行了详细的表征。在底物结合区I（SRBI）中，缬氨酸193取代脯氨酸，使pH值从最适值向中性偏移了1个单位。然后，对该区域的许多氨基酸残基进行了位点特异性突变，随后对其pH最优、叠氮化物敏感性和热稳定性进行了表征。V193P和P190I的pH最优值明显升高，但没有改变其他酶的性质。基于计算机图形模拟推导出的ASOM结构，Glu192与Arg309形成离子对。用脯氨酸替代Val193可能会破坏这种离子对，并可能在Glu192和Arg309之间形成新的稳定离子对。特异性口袋构象的改变影响了酶催化的最佳pH值。

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.