来源于软化芽孢杆菌的环糊精葡萄糖基转移酶在毕赤酵母和枯草杆菌中的表达

Expression of Paenibacillus macerans cyclodextrin glycosyltransferase in Pichia pastoris and Bacillus subtilis

摘要:

通过PCR扩增软化芽孢杆菌a-CGT酶基因, 将基因片段分别克隆到毕赤酵母表达载体pPIC9K和大肠杆菌-枯草杆菌穿梭载体pMA5中, 分别转化毕赤酵母KM71和枯草杆菌WB600。结果表明, 重组毕赤酵母发酵上清液中a-CGT酶活性仅0.2 U/mL, 重组枯草杆菌产酶达到1.9 U/mL。对重组枯草杆菌发酵条件进行了优化, 当以TB为出发培养基, 初始pH 6.5, 温度为37oC时, 摇瓶培养24 h后a-CGT酶环化活性达到4.5 U/mL (水解活性为3200 IU/mL), 是野生菌株软化芽孢杆菌表达量的9.8倍。

Abstract:

The cgt gene was isolated from Paenibacillus macerans by PCR amplification and was inserted into vectors of pPIC9K and pMA5. The recombinant vectors were transformed to Pichia pastoris KM71 and Bacillus subtilis WB600, respectively. The results showed that a-CGTase activity in the culture media of recombinant P. pastoris was only 0.2 U/mL, while it was 1.9 U/mL in recombinant B. subtilis. In addition, we optimized the culture conditions of the recombinant B. subtilis strain. After cultivation at 37oC for 24 h with shake flask, the CGTase forming activity in culture media reached to 4.5 U/mL (hydrolysis activity was 3200 IU/mL), which is 9.8-fold to that of the original strain P. macerans.