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。
A codon optimized thermostable pullunase gene（Bn pulB） was synthesized and cloned behind an artificial tandom promter Pga2. The synthetic cassette was used to generate a suicide plasmid pGE-BPB; transformation of the plasmid into Bacillus subtilis 168 led to a pullulanase producing recombinant strain CH-1 with native nprE deleted. Initially,30.3 U/mL of extracellular pullulanase was detected by CH-1 on regular medium supplemented with glucose,up to 268 U/mL of enzyme was detected by fermentation at 32 ℃ in medium with initial pH 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.
出处
《食品工业科技》
CAS
CSCD
北大核心
2015年第20期214-219,共6页
Science and Technology of Food Industry
基金
上海市长三角科技联合攻关领域项目(15295810600)
关键词
普鲁兰酶
枯草芽胞杆菌
基因合成
串联启动子
发酵优化
pullulanase
Bacillus subtilis
synthetic operon
tandem promoter
fermentation optimization
