米曲霉酸性蛋白酶Ap的结构分析

Structural Analysis of Acid Protease( Ap) from Aspergillus oryzae

米曲霉(Aspergillus oryzae)酸性蛋白酶与部分商品化的蛋白酶相比,具有对大豆蛋白水解效率高且水解产物苦味弱的特性。作者采用RT-PCR技术克隆获得酸性蛋白酶(Ap)基因,结合生物信息学手段,对Ap进行分析与预测。结果表明:Ap基因对密码子使用频率具有偏好性,特别是第3位密码子对GC碱基的使用频率高,达到74%;编码404个氨基酸残基。Ap属于胞外天冬氨酸蛋白酶。以海枣曲霉(A.phoenicis)的酸性蛋白酶(PDB code:1IBQ)作为模板同源建模结果可知,Ap分子至少能与2个Zn2+结合,内含1个二硫键、163个氢键、35个盐碱。该酶与1IBQ相比空间结构总体相似,部分重要位点(如flap环、转角和ψ-loops)的氨基酸残基较为保守。

Compared with some commercial proteases,acid protease（Ap） from Aspergillus oryzae has high hydrolysis efficiency and produces soy hydrosates with weaker bitterness. In order to uncover the structural characteristics,Ap gene from A. oryzae was cloned by RT-PCR technology,and then was analyzed with bioinformatics technology. Results showed that Ap gene had a preference for codon usage,especially for the third codon with the 74% usage frequency of GC base,and encoded 404 amino acid residues. Ap was an extracellular aspartic proteinase. According to the structure obtained by homology modeling with acid protease from A. phoenicis（PDB code：1IBQ）,Ap was found to possess at least two Zn2＋binding sites,one disulfide bond,163 hydrogen bonds,35 salt bonds,and two solvent accessible surfaces of D33（4.7849魡2） and D215（3.5941魡2）. Though the spatial structures of Ap and 1IBQ were similar,residues of some important sites（such as flap ring,angle and ψ-loops） were conservative.