摘要
Taq DNA聚合酶是PCR技术中广泛应用的耐热酶。为提高重组大肠杆菌生产Taq DNA聚合酶的产量,以Taq DNA聚合酶产量和酶的比活力为考察指标,通过单因素实验优化产生TaqDNA聚合酶含量的单因素:发酵温度、pH值、IPTG诱导剂用量、诱导时间、接种量,结果表明:发酵温度、pH值、IPTG诱导剂浓度,对该基因工程菌发酵产Taq DNA聚合酶的比酶活力有显著影响,而诱导培养时间、接种量对产Taq DNA聚合酶的比酶活力影响不显著。故选择发酵温度、pH值、IPTG诱导剂浓度作为响应面的三个因素优化重组大肠杆菌生产Taq DNA聚合酶发酵工艺,响应面分析结果显示对酶的比活力显著性顺序为:IPTG诱导剂浓度>发酵温度>pH值,最佳发酵工艺参数为:发酵温度37.2℃,pH值为7.5,IPTG诱导剂浓度为0.800 mmol/L,在此条件下发酵,Taq DNA聚合酶比活力达到(43.822±0.878)kU/mg。
Taq DNA polymerase is a thermostable enzyme and widely used in PCR technology.In order to improve the production of Taq DNA polymerase by recombinant Escherichia coli,in this study the enzyme yield and specific activity of the enzyme were taken as the inspection indicators.The single factors producing Taq DNA polymerase content were optimized through single factor test:fermentation temperature,pH,amount of IPTG inducer,induction time,and inoculation amount.The results showed that fermentation temperature,pH,concentration of IPTG inducer had a significant impact on the specific enzyme activity of Taq DNA polymerase produced by the genetic engineering strain,while induction culture time and inoculation amount had no significant impact on the specific enzyme activity of Taq DNA polymerase produced by the genetic engineering strain.So fermentation temperature,pH and IPTG inducer concentration were selected as three factors of response surface to optimize the fermentation process of recombinant Escherichia coli for Taq DNA polymerase production.The response surface analysis results showed and that the order of significance for the specific activity of enzymes were:IPTG inducer concentration>fermentation temperature>pH,and the optimal fermentation process parameters were:fermentation temperature 37.2℃,pH 7.5,IPTG inducer concentration 0.800 mmol/L.Under this condition,the specific activity of Taq DNA polymerase reached(43.822±0.878)kU/mg.
作者
肖云
陈洁
XIAO Yun;CHEN Jie(College of Bioengineering,Wuhan Polytechnic,Wuhan,Hubei 430072,China)
出处
《粮油食品科技》
CAS
CSCD
2023年第4期154-161,共8页
Science and Technology of Cereals,Oils and Foods
基金
武汉职业技术学院校级项目(2022YK035)。
关键词
重组大肠杆菌
Taq
DNA聚合酶
酶比活力
发酵工艺
响应面分析法
recombinant Escherichia coli
Taq DNA polymerase
specific activity of enzyme
fermentation process
response surface methodology
