摘要
【目的】构建分泌表达α-环糊精葡萄糖基转移酶(α-CGTase)的重组枯草芽胞杆菌,实现α-CGTase的安全高效表达。【方法】通过PCR法扩增嗜热地芽胞杆菌α-CGTase基因,运用EcoRI/Xho I分别对α-CGTase基因及pBES进行双酶切,然后将该基因片段插入到大肠杆菌-枯草芽胞杆菌穿梭载体pBES中,再电转化法转化枯草芽胞杆菌RIK1285;对重组枯草芽胞杆菌B.subtilis RIK1285/pBE-CGT发酵条件进行探索。【结果】(1)利用发酵培养基分泌表达α-CGTase,重组枯草芽胞杆菌工程菌B.subtilis RIK1285/pBE-CGT发酵上清液产酶达到2.9U·mL~(-1)。(2)TB为最适发酵培养基(配方:甘油0.5%,蛋白胨1.2%,酵母粉2.4%,K_2HPO_4 1.64%,KH_2PO_40.23%);在初始pH 6.5,温度为37℃下,摇瓶发酵培养24h后,α-环糊精酶的环化活性达到5.3U·mL~(-1),是野生菌株嗜热地芽胞杆菌(0.66U·mL~(-1))的8倍。【结论】成功构建了枯草芽胞杆菌B.subtilis RIK1285/pBE-CGT工程菌,并确定其最适发酵培养基和培养条件。
【Objective】To construct a vector for expressingα-cyclodextrin glycosyltransferase(α-CGTase)gene in Bacillus subtilis.【Method】Theα-CGTase gene from Gebacillius sp.CHB1 was amplified by PCR.The EcoRIdigested pBES and Xho I-digested α-CGT gene were connected and transformed into B.subtilis RIK1285.Subsequently,fermentation of the recombinant B.subtilis RIK1285/pBE-CGT was optimized.【Result】(1)Theα-CGTase gene was expressed in a fermentation medium to show an enzymatic activity of 2.9 U·mL~(-1) by B.subtilis RIK1285/pBE-CGT.(2)Medium TB with the formula of 0.5% glycerol,1.2% peptone,2.4% yeast extract,1.64% K_2HPO_4,and 0.23% KH_2PO_4 was found to be optimal for the fermentation.After fermentation in TB at37℃ for 24 h,theα-CGTase activity reached 5.3 U·mL~(-1),which was 8-fold of what the wild Gebacilliussp.CHB1 could generate.【Conclusion】The engineered B.subtilis RIK1285/pBE-CGT was successfully obtained and the fermentation process optimized.
引文
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