摘要
为达到褐色嗜热裂孢菌葡萄糖异构酶在大肠杆菌中的过量表达,褐色嗜热裂孢菌葡萄糖异构酶的编码基因被植入含有T7强启动子的表达载体p ET-24a(+)中,并转入重组大肠杆菌BL21(DE3)进行表达,于3L发酵罐中采用两阶段补料策略进行高密度发酵产酶。在诱导前阶段,设定比生长速率μset=0.25,补料流加速率以指数方式增长;在诱导阶段,根据诱导后培养基中甘油残留量和乙酸的积累量,补料流加速率以梯度方式下降。在此流加策略下进一步考察影响发酵产酶的诱导强度、诱导节点、诱导温度和复合氮源的添加量等因素。最终根据两阶段流加策略,在乳糖流加速率0.2 g/(L·h),诱导时菌浓(DCW)30 g/L,诱导温度25℃和适量添加复合氮源的条件下,1 m L发酵液的菌体细胞内重组GIase产量达到124 U,这是通过菌体发酵产GIase获得的最高产量。
This work aims to achieve the overproduction of Thermobifida fusca glucose isomerase（GIase） with recombinant Escherichia coli by a two-stage nutrient feeding strategy. First, the GIase coding gene from Thermobifida fusca WSH03-11 was expressed in E. coli BL21（DE3） under the strong inducible T7 promoter of the pET-24 a（＋） vector.Then a two-stage feeding strategy was tried in a 3 L fermenter, where cultivation was conducted at a controlled specific growth rate（μset= 0.25） during pre-induction phase, followed by a gradient-decreasing feeding rate at induction phase.And then induction temperature, inducer lactose flow rate, induction point and the amount of complex nitrogen source added in feeding solutions on cell growth and GIase production was investigated. Finally, when flow rate of lactose at 0.2 g/（L·h）, induced point at a cell concentration of 30 g/L, induced temperature at 25 °C, and moderate complex nitrogen supplementation, GIase yield of 1 m L fermented liquid could reach 124 U, which is the highest GIase production by recombinant E. coli expression system.
出处
《中国食品学报》
EI
CAS
CSCD
北大核心
2018年第2期79-87,共9页
Journal of Chinese Institute Of Food Science and Technology
基金
国家自然科学基金面上项目(81573536)
安徽省教育厅自然科学基金重点项目(KJ2013A264
KJ2015A454)
安徽高校优秀青年人才支持计划项目(gxyq2017057)
安徽省高校自然科学基金重点项目(WXZR201616)
关键词
褐色嗜热裂孢菌葡萄糖异构酶
两阶段流加策略
高密度发酵
诱导条件优化
Thermobiftda fusca glucose isomerase
two-stage nutrient feeding strategy
high density fermentation
optimization of induction conditions
