环状芽孢杆菌α-羟基-γ-氨丁酰酰化酶基因的克隆和表达

Cloning and Expression of α-Hydroxy-γ-Aminobutyl Acylase Gene of Bacillus circulans NRRL-B3312

本文报道了以质粒pUB110为载体,以枯草芽孢杆菌(Bacillus subtilis 168)作为受体菌,对丁酰苷菌素产生菌(Bacillus circulans NRRL-B3312)总DNA进行了鸟枪克隆,在所获得的转化子中,No.733转化子经薄层层析,生物显迹和质谱分析表明,它具有将卡那霉素A生物转化成为丁胺卡那霉素的能力,说明该转化子所含重组质粒pUBC733的插入片段中含有a-羟基-r-氨丁酰(HABA)酰化酶基因,HABA酰化酶基因已经在枯草芽孢杆菌中获得了克隆和表达。该重组质粒分子量为7.3kb,插入片段为2.8kb,经Southern分子杂交确证此片段确来源于环状芽孢杆菌,已构建了该质粒限制性内切酶图谱。

With shot-gun cloning strategy, we used pUB110 plasmid as a vactor to clone DNA fragment of Bacillus circulans NRRL-B3312, which is butirosin producer, into Bacllus subtilis 168. Among the transfonnants, the results of TLC, bioautography and FAB mass, spectrum analysis for the bioconversion product of No.733 tramforment showed that this transformant could transform kanamycin into amikacin. According to these results, the HABA acylase pene locates on the insert fragment of pU'BC733 plasmid harbouring No.733 transformant. We can confirm that the HABA acylase gene was cloned and expressed in B. subtilis 168. Molecular weight of-pUBC733 is 7.3kb. Southern hybridization demonstrated that the 2.8kb inserted fragment of this plasmid originated from B. circulans NRRL-B3312. The restriction map of pUBC733 plasmid was constructed. ,