普鲁兰酶基因在毕赤酵母中组成型表达及定点突变研究

合成Bacillus acidopullulyticus的全长普鲁兰酶基因并在毕赤酵母X-33中进行组成型外分泌表达,重组酶的最适作用温度为60℃,最适作用pH值为4.5~5.0,酶比活力为2.0 U/mg.采用重叠延伸PCR方法对普鲁兰酶基因进行定点突变,实验结果表明,625、626位点Ala、Leu氨基酸突变为Leu、Tyr氨基酸后,该酶的催化效率有所降低,而Gln487Ala的突变对催化效率没有较大的影响.该研究结果为探究关键氨基酸区域对催化效率的影响提供了一定的理论和实验基础.

The constitutive expression of gene encoding pullulanase in Pichia pastoris and site-directed mutagenesis

The full-length gene pull3 encoding pullulanase from Bacillus acidopuUulyticus was synthetized. And the secretion expres- sion vector pGAPZcL A-pull3 was induced into Pichia pastoris X33 by electrotransformation. The optinmm reaction temperature and pH of recombinant pullulanasc, which has the specific activity of 2.0 U/mg, were found to be 60~C and 4. 5 - 5.0, respectively. Further- more, site-directed mutagenesis of pull3 by overlap extension PCR was introduced to study the structure and function of enzymes. A decrease of the specific activity of this pullulanase was seen upon the replacement of Ala Leu （625 -626） by Leu Tyr. However, the mutations Gln487Ala showed the same specific activity with the wild-type pullulanase. The results provided a theoretical basis for ex- ploring the roles of key active-site residues in pullulanase.