血红蛋白基因在枯草芽孢杆菌中的表达及其作用的研究

Expression and Roles of Hemoglobin Gene in Bacillus subtilis

革兰氏阴性菌Ｖｉｔｒｅｏｓｃｉｌｌａ的血红蛋白与氧结合力强，能降低该菌需氧量。将该血红蛋白的结构基因（Ｖｇｂ）连接到枯草杆菌质粒ｐＡＫ４的β－内酰胺酶基因（ｂｌａ）启动子下（框架正确），构建了重组质粒ｐＡＶ，并将此质粒先转化至枯草杆菌ＤＢ １０４，继而又转化到枯草杆菌碱性蛋白酶工程菌Ｇ３３１和产木聚糖酶枯草杆菌Ｂ５３。经酶切和电泳分析显示转化株的质粒ＤＮＡ含有大小与 Ｖｇｂ相同的电泳带，又经非放射性同位素标记杂交实验，证明为含Ｖｇｂ基因的克隆，并采用一氧化碳鼓泡法测出血红蛋白的表达。在试管和三角瓶发酵实验中，在同一培养条件下碱性蛋白酶活性和木聚糖酶活性有所提高，为获得枯草杆菌发酵中降低需氧量，提高产量研究开辟新途径。

Hemoglobin of the Gram--negative bacterium Vitreoscilla can bind oxygen strongly and reduce the oxygen requirement of the bacteritun. A recombinant plasmid pAV was constructed by inserting the hemoglobin gene (Vgb) to the downstream of the promoter of --lactase gene of the B. subtilis plasmid pAK4. The plasmid pAv was transferred into strain DB104, G331 (a subtilisin--producing B. subtilis) and B53 (a xylanase-producing B. subtilis). The subclones and transfer of the Vgb gene into pAK4 were detected and confirmed by Dot blotting and electrophoresis analysis. The expression of homoglobin was measured by recording CO-difference spectra after bubbling CO into the sample cuvette. The production of subtilisin and xylanase was increased in the fermentation of the Vgb-expressing strains. These results provided a new way to reduce the oxygen requirement and to increase the biomass production in the fermentation industry.