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

作　　者：王凤寰[1] 孟青青[1] 封棣[1] 杨建国[2] 汪兵 

机构地区：[1]北京工商大学食品学院、食品添加剂与配料北京高校工程研究中心、北京市食品风味化学重点实验室,北京100048 [2]北京化工大学生命科学与技术学院,北京100029 [3]北京中天诺亚酶制剂有限公司,北京102629

出　　处：《生物技术通报》2012年第7期119-125,共7页Biotechnology Bulletin

基　　金：国家自然科学基金项目(20906003)

摘　　要：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.

关 键 词：黑曲霉 硫磺矿硫化叶菌 嗜热糖化酶 同源重组 热稳定性 

分 类 号：Q78[生物学—分子生物学]