嗜热糖化酶基因在重组黑曲霉工程菌中的表达及酶学性质初步研究

Expression of Thermostable Glucoamylase Gene in Aspergillus niger and Its Characterization

为了提高糖化酶的耐热性能,降低淀粉糖化发酵工艺的生产成本,构建了同源整合载体pEasy-glaAdir以及pEasyssg,将黑曲霉(Aspergillus niger)的糖化酶基因(glaA)灭活,并将硫磺矿硫化叶菌(Sulfolobus solfataricus)的嗜热糖化酶基因(ssg)插入到黑曲霉基因组中,筛选得到表达嗜热糖化酶的重组黑曲霉工程菌(A.nigerWW1)。重组菌的发酵结果显示,嗜热糖化酶在黑曲霉中得到了分泌表达,发酵液酶活达到3 030 U/mL。重组嗜热糖化酶的最适反应温度为90℃,最适pH为6.0,该酶具有较高的热稳定性,在80℃时的半衰期在60 min以上,具有良好的工业应用前景。

It was to improve the glucoamylase thermostability and reduce the cost of industrial fermentation, integrative vectors pEasyglaA^dir and pEasy-ssg were constructed and transformed into Aspergillus niger CICC 2204. The native glaA gene was interrupted and the Sulfolobus solfataricus glaA gene （ ssg ） encoding the thermostable glucoamylase was integrated into the chromosome of A. niger, which yielded a thermostable glucoamylase producer. The positive transformant showed great secretive expression of SSG and the enzyme activity reached 3 030 U/mL. The recombinant thermostable glucoamylase exhibited optimum catalytic activity at 90℃ and pH6.0, respectively, and remained 50% of its original activity after 60 min at 80℃. It was more thermostable than that of native GLA.