利用指数流加补料高密度发酵产β-呋喃果糖苷酶重组大肠杆菌

High density fermentation for beta-fructofuranosidase production by recombinant Escherichia coli BL21(DE3) using exponential fed-batch culture strategy

摘要:

【目的】对重组大肠杆菌BL21(DE3)/pET22b-β-ffase进行高密度发酵产β-呋喃果糖苷酶工艺研究。【方法】比较溶氧反馈补料和指数流加补料对重组菌发酵产酶的影响，对不同比生长速率和诱导时机进行优化。【结果】确定了双阶段指数流加过程中重组菌生长的比生长速率，分别控制诱导前期比生长速率为0.20 h?1，诱导后期比生长速率为0.13 h?1，诱导时机为指数中期。获得细胞干重约为51 g/L，最高酶活达到1.79×105 U/L，单位菌体产酶量为3 510 U/g，单位产酶速率达到3.58×104 U/(L·h)，生物量、单位菌体产酶量和产酶速率分别是指数流加未优化前的1.8、1.7和3.0倍。【结论】双阶段指数流加补料工艺能有效提高β-呋喃果糖苷酶的产酶量，为β-呋喃果糖苷酶的进一步工业化奠定基础。

Abstract:

[Objective] To study the high density fermentation process for beta-fructofuranosidase production by recombinant Escherichia coli BL21(DE3)/pET22b-β-ffase. [Methods] we compared the influence of the dissolved oxygen feedback control and the exponential fed-batch culture on the β-FFase production of the Escherichia coli BL21(DE3)/pET22b-β-ffase fermentation, and the specific growth rate and induction time were optimized. [Results] This study confirmed the specific growth rate during the dual-stage specific growth rate controlled exponential fed-batch culture. Specific growth rate were controlled at 0.20 h?1 and 0.13 h?1 in pre-induction phase and late phase after induction respectively, the express system can be induced at mid-logarithmic phase. Finally, cell dry weight reached up to 51 g/L, the peak of activity attained at 1.79×105 U/L, the unit cell enzyme activity leaped to 3 510 U/g, and the enzyme production rate reached up to 3.58×104 U/(L·h). The biomass, the unit cell enzyme activity and the enzyme production rate have been strikingly improved, which were 1.8-fold, 1.7-fold and 3.0-fold compared with values of pre-exponential feeding culture without optimization. [Conclusion] Dual-stage specific growth rate controlled exponential fed-batch culture significantly contributes to improve the enzyme β-fructofuranosidase production, and also lays the foundation for the further industrialization of the enzyme.