Ullah A H, Sethumadhavan K, Lei X G, Mullaney E J
Southern Regional Research Center, New Orleans, Louisiana 70124, USA.
Biochem Biophys Res Commun. 2000 Aug 28;275(2):279-85. doi: 10.1006/bbrc.2000.3271.
The gene for Aspergillus fumigatus phytase (phyA) was cloned and expressed in Pichia pastoris. The enzyme expressed was purified to near homogeneity using sequential ion-exchange chromatography and was characterized biochemically. Although A. fumigatus phytase shows 66.2% sequence homology with A. ficuum phytase, the most widely studied enzyme, the cloned phytase showed identical molecular weight and temperature optima profile to the benchmark phytase. The pH profile of activity and kinetic parameters, however, differed from A. ficuum phytase. The cloned enzyme contains the septapeptide RHGARYP motif, which is also identical to the active site motif of A. ficuum phytase. Chemical probing of the active site Arg residues using both cyclohexanedione and phenylglyoxal resulted in the inactivation of phytase. The cloned A. fumigatus phytase, however, was more resistant to phenylglyoxal-induced inactivation. Both cloned A. fumigatus and A. ficuum phytases were identically affected by cyclohexanedione. Both the thermal characterization data and kinetic parameters of cloned and expressed A. fumigatus phytase indicate that this biocatalyst is not superior to the benchmark enzyme. The sequence difference between A. fumigatus and A. ficuum phytase may explain why the former enzyme catalyzes poorly compared to the benchmark enzyme. In addition, differential sensitivity toward the Arg modifier, phenylglyoxal, indicates a different chemical environment at the active site for each of the phytases.
烟曲霉植酸酶（phyA）基因被克隆并在毕赤酵母中表达。表达的酶通过连续离子交换色谱法纯化至接近均一，并进行了生化特性分析。尽管烟曲霉植酸酶与研究最为广泛的泡盛曲霉植酸酶有66.2%的序列同源性，但克隆的植酸酶与基准植酸酶显示出相同的分子量和最适温度曲线。然而，活性的pH曲线和动力学参数与泡盛曲霉植酸酶不同。克隆的酶含有七肽RHGARYP基序，这也与泡盛曲霉植酸酶的活性位点基序相同。使用环己二酮和苯乙二醛对活性位点精氨酸残基进行化学探测导致植酸酶失活。然而，克隆的烟曲霉植酸酶对苯乙二醛诱导的失活更具抗性。克隆的烟曲霉和泡盛曲霉植酸酶对环己二酮的影响相同。克隆和表达的烟曲霉植酸酶的热特性数据和动力学参数均表明，这种生物催化剂并不优于基准酶。烟曲霉和泡盛曲霉植酸酶之间的序列差异可能解释了为什么前者酶与基准酶相比催化性能较差。此外，对精氨酸修饰剂苯乙二醛的不同敏感性表明，每种植酸酶活性位点的化学环境不同。
