嗜酸芽孢杆菌普鲁兰酶在大肠杆菌中的表达及发酵优化

Expression of Bacillus acidopullulyticus pullulanase in Escherichia coli and its optimization of fermentation

为了在3L发酵罐水平上提高重组菌E.coli BL 21(DE3)/pET20b(+)-BapulA的普鲁兰酶的表达量和胞外分泌,将嗜酸芽孢杆菌普鲁兰酶(Bacillus acidopullulyticus)在大肠杆菌中进行重组表达,并在3L发酵罐水平上对重组菌进行发酵优化,考察了发酵条件对重组酶表达的影响。重组菌发酵的最佳条件为:发酵前期菌体生长温度和pH分别为30℃和7.0,当菌体浓度OD_(600)达到50时,发酵温度降低至25℃,此时调节pH为6.2,同时开始流加乳糖[0.4g/(L·h)]进行诱导;在发酵过程中,当菌体浓度OD_(600)依次达到15,45,75时,分别加入浓度为1.5g/L的甘氨酸,当菌体OD600为105时,再加入浓度为3g/L的甘氨酸。发酵结束后普鲁兰酶的胞外酶活达659.0U/mL,最高总酶活达1 910.1U/mL,与摇瓶的初始总酶活(41.1U/mL)和胞外酶活(6.5U/mL)相比,分别提高了45.4倍和100倍。

To increase expression and extracellular secretion of pullulanase of the recombinant strain E.coli BL21（DE3）/pET20 b（＋）-BapulA at 3 L fermentor level. Expression of recombinant pullulanase in E.coli and further optimization of fermentation at the3 Lfermenter Firstly.The effects were investigaged,including the fermentation temperature,pH,IPTG concentration,induction time and glycine concentration on the expression of recombinant pullulanase.The optimum fermentation conditions were showed as followed：the growth temperature and pH of the pre-induction cells were 30 ℃ and 7.0,respectively.When the cell density reached 50 OD_（600）,the fermentation temperature was shifted to 25 ℃,and the pH was adjusted to 6.2 with lactose flow rates of 0.4 g/（L·h）.During the fermentation process,glycine was added at the concentration of 1.5 g/L when the cell density of OD_（600） reached 15,45 and 75,respectively.When OD600 reached 105,glycine was added at a concentration of 3 g/L.The optimal total and extracellular pullulanase activity were 1 910.1 U/mL and 659.0 U/mL,respectively,which represent 45.4 and 100-fold compared with those observed under initial conditions.