Gu Wei-Na, Yang Pei-Long, Wang Ya-Ru, Luo Hui-Ying, Meng Kun, Wu Ning-Feng, Yao Bin, Fan Yun-Liu
Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Sheng Wu Gong Cheng Xue Bao. 2007 Mar;23(2):273-7.
Aspergillus fumigatus wild-type phytase has many favorable properties, such as a good thermorstability and a broad pH optimum. However, the specific activity of the enzyme is relative low. A. fumigatus Q23L phytase resulted in a remarkable increase in specific activity around pH4.5 - 7.0, but the pH stability of Q23L was lower than A. fumigatus wild-type phytase. To increase the pH stability of Q23L, the mutant Q23LG272E was constructed by site-directed mutagenesis with PCR. The gene of A. fumigatus wild-type phytase and the mutant genes encoding the Q23LG272E and the Q23L were correctly expressed in Pichia pastoris GS115. Enzymes were purified and their enzymatic properties were determined. The results revealed that the specific activity of the Q23L improved remarkably, which increased from 51 u/mg of the wild type to 109 u/mg at pH5.5. Meanwhile, the pH stability of Q23L, decreased evidently, especially from pH3.0 to pH4.0.The pH stability of Q23LG272E in pH3.0 - 4.5 and pH6.5 - 7.0 has been improved compared with Q23L. The specific activity of Q23LG272E basically maintained at the level of Q23L. Analysis of 3-D structure and sequence similarity were used to reveal the presumable factors influencing the enzymatic properties of Q23LG272E, and discussion for the relationship between structure and function of phytase was given.
烟曲霉野生型植酸酶具有许多优良特性，如良好的热稳定性和较宽的最适pH值范围。然而，该酶的比活性相对较低。烟曲霉Q23L植酸酶在pH4.5 - 7.0左右比活性显著增加，但Q23L的pH稳定性低于烟曲霉野生型植酸酶。为提高Q23L的pH稳定性，通过PCR定点诱变构建了突变体Q23LG272E。烟曲霉野生型植酸酶基因以及编码Q23LG272E和Q23L的突变基因在毕赤酵母GS115中正确表达。对酶进行了纯化并测定了其酶学性质。结果表明，Q23L的比活性显著提高，在pH5.5时从野生型的51 U/mg增加到109 U/mg。同时，Q23L的pH稳定性明显下降，尤其是在pH3.0至pH4.0之间。与Q23L相比，Q23LG272E在pH3.0 - 4.5和pH6.5 - 7.0的pH稳定性有所提高。Q23LG272E的比活性基本维持在Q23L的水平。利用三维结构分析和序列相似性来揭示影响Q23LG272E酶学性质的可能因素，并对植酸酶的结构与功能关系进行了讨论。
