普鲁兰酶在需钠弧菌中的分泌表达与发酵优化

Secretory expression and fermentation optimization for extracellular production of pullulanase in Vibrio natriegens

摘要:

普鲁兰酶是一种淀粉脱支酶，因其分子量较大，胞外分泌表达难度较高。需钠弧菌(Vibrio natriegens)是一种新型的蛋白表达宿主，拥有高效的蛋白合成效率。本研究使用基因组整合T7 RNA聚合酶表达框的V.natriegens VnDX为宿主，构建了产全长普鲁兰酶PulA及其截短突变体PulN2的重组需钠弧菌，分析了信号肽、发酵温度、诱导剂浓度、甘氨酸浓度及发酵时间等条件对产酶的影响，并且对比了2种普鲁兰酶在V.natriegens VnDX与大肠杆菌(Escherichia coli) BL21(DE3)中的胞外产酶能力。研究结果显示，普鲁兰酶PulA和PulN2在V.natriegens VnDX中的胞外酶活为61.6 U/mL和64.3 U/mL，分别为E.coli BL21(DE3)最大酶活力的110%和62%。上述结果表明V.natriegens VnDX可以分泌表达大分子量的全长普鲁兰酶PulA，本研究可为其他大分子量蛋白在V.natriegens VnDX中的分泌表达提供参考和借鉴。

关键词:需钠弧菌;普鲁兰酶;分泌表达;发酵条件优化

Abstract:

Pullulanase is a starch debranching enzyme, which is difficult in secretory expression due to its large molecular weight. Vibrio natriegens is a novel expression host with excellent efficiency in protein synthesis. In this study, we achieved secretory expression of the full-length pullulanase PulA and its truncated mutant PulN2 using V. natriegens VnDX strain. Subsequently, we investigated the effects of signal peptide, fermentation temperature, inducer concentration, glycine concentration and fermentation time on the secretory expression. Moreover, the extracellular enzyme activities of the two pullulanases produced in V. natriegens VnDX and E. coli BL21(DE3) were compared. The highest extracellular enzyme activity of PulA and PulN2 in V. natriegens VnDX were 61.6 U/mL and 64.3 U/mL, which were 110% and 62% that of those in E. coli BL21(DE3), respectively. The results indicated that V. natriegens VnDX can be used for secretory expression of the full-length PulA with large molecular weight, which may provide a reference for the secretory expression of other large molecular weight proteins in V. natriegens VnDX.

Key words:Vibrio natriegens;pullulanase;secretory expression;fermentation optimization