Construction of a thermostable pullulanase producing bacillus strain and its fermentation studies
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摘要: 将长野芽胞杆菌的普鲁兰酶基因经密码子优化后,组建了人工合成的二联启动子Pga2,并将它克隆到枯草芽胞杆菌穿梭质粒p MK4-BPB以及自杀质粒p GE-BPB中;经转化和筛选获得了中性蛋白酶基因npr E被敲除的普鲁兰酶生产菌株CH-1;该重组菌在基础培养基中所产普鲁兰酶的酶活达到30.3 U/m L;经过对培养基组分及发酵条件（培养温度、起始p H,起始接种量等）进行优化,确定了发酵的最适碳源为45 g/L的蔗糖,氮源为60 g/L的麸皮+豆粕时,设定初始培养基的p H为6.2,在培养温度为32℃时进行发酵,CH-1发酵产重组普鲁兰酶酶活高达268 U/m L。Abstract: A codon optimized thermostable pullunase gene(Bn pul B) was synthesized and cloned behind an artificial tandom promter Pga2. The synthetic cassette was used to generate a suicide plasmid p GE-BPB;transformation of the plasmid into Bacillus subtilis 168 led to a pullulanase producing recombinant strain CH-1with native npr E deleted. Initially,30.3 U/m L of extracellular pullulanase was detected by CH-1 on regular medium supplemented with glucose,up to 268 U/m L of enzyme was detected by fermentation at 32 ℃ in medium with initial p H at 6.2,using 45 g/L of sucrose as carbon source and 60 g/L of wheat bran plus bean pulp as nitrogen source.
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Keywords:
- pullulanase /
- Bacillus subtilis /
- synthetic operon /
- tandem promoter /
- fermentation optimization
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