Aspergillus fumigatus来源植酸酶的Q23L和Q23LG272E突变研究

Mutation Research on Q23L and Q23LG272E in Phytase Derivated from Aspergillus fumigatus

Aspergillus fumigatus来源的植酸酶具有热稳定性好、pH作用范围广的优点,但其比活性很低。设计的植酸酶Q23L突变能在pH4.5～7.0范围内大幅提高比活性,但pH稳定性却显著下降,为了进一步改良Q23L的pH稳定性,在Q23L分子上加入了G272E突变。将原酶、突变酶Q23L和突变酶Q23LG272E分别在毕赤酵母GS115中表达,表达酶经纯化后进行酶学性质比较分析,结果表明:突变酶Q23L的比活性比原酶显著提高,在pH5.5比活性由51u/mg提高到109u/mg,但其pH稳定性,尤其是在pH3.0～4.0酸性条件下的稳定性却显著降低,低于80%。突变酶Q23LG272E在pH3.0～4.5和pH6.5～7.0时的稳定性比Q23L有所提高,恢复到原酶的水平,而比活性基本维持在Q23L的水平。通过一级序列和三维结构比较,分析了可能影响Q23LG272E酶学性质的因素,为进一步研究植酸酶的结构与功能提供了材料。

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 51u/mg of the wild type to 109u/mg at pHS.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.