黄杆菌肝素酶Ⅱ重组菌培养条件的优化及高密度发酵

Culture conditions optimization and high cell density fermentation of recombinant bacteria producing heparinaseⅡ from Flavobacterium heparinum

摘要:

黄杆菌肝素酶Ⅱ (HepⅡ) 是一类可特异性切割肝素、乙酰肝素类分子内连接键的酶。文中对黄杆菌肝素酶Ⅱ重组菌的诱导时机、诱导剂添加量、诱导温度、诱导时间等诱导产酶条件进行优化。经过优化最佳摇瓶发酵产酶条件为：37 ℃ 培养重组菌至对数生长前期，添加诱导剂IPTG至终浓度为0.3 g/L，20 ℃下诱导10 h，酶活达到最高，为570 U/L。在此基础上通过发酵罐高密度培养手段将菌体浓度OD600进一步提高到98，酶活大幅度提高到9 436 U/L，该研究结果为HepⅡ的工业化生产与应用奠定了良好的基础。

Abstract:

HeparinaseⅡ (HepⅡ) from Flavobacterium heparinum is an enzyme that could specifically cleave certain sequence of heparin and heparan sulfate. In this work, fermentation conditions of recombinant heparinase Ⅱ (His-Hep Ⅱ) producing bacteria were optimized, including initial induction time, inducer (IPTG) concentration, induction temperature and induction time. The optimum conditions were as follows: cultivating recombinant bacteria to exponential prophase under 37 ℃, then adding IPTG to a final concentration of 0.3 g/L, finally cultivating recombinant bacteria under 20 ℃ for 10 h. The total crude enzyme activity reached 570 U/L. Based on these results, high cell density fermentation of recombinant bacteria was studied. The final OD600 could reach 98 and the total crude enzyme activity of His-Hep Ⅱ increased to 9 436 U/L.