极端嗜热酸性α-淀粉酶PFA在枯草芽孢杆菌中的高效分泌表达

Efficient Secretory Expression of Hyperthermoacidophilic α-Amylase PFA in Bacillus subtilis WB600

为探索极端嗜热酸性α-淀粉酶PFA在枯草芽孢杆菌(Bacillus subtilis WB600)中的高效分泌表达条件,对来源于枯草芽孢杆菌的9种Sec分泌途径信号肽进行筛选,结果显示信号肽Yfk N的引导分泌效率最高。在此基础上,为进一步优化PFA的分泌表达,对信号肽Yfk N的Ile3和Gln4进行饱和突变,并比较不同突变体的引导分泌效率。结果表明突变体I3G/Q4R的引导分泌效率最高,重组枯草芽孢杆菌的胞外α-淀粉酶活力高达715 U/m L。重组α-淀粉酶PFA的最适反应p H值为5.0,最适反应温度为100℃,于100℃的半衰期长达13 h,并且不依赖于Ca^(2+)。结果表明,采用信号肽I3G/Q4R,极端嗜热酸性α-淀粉酶PFA能够在枯草芽孢杆菌中高效分泌表达,这有利于其在淀粉液化工艺中的应用。

In this study,we screened 9 secretory(Sec)pathway signal peptides for the production of recombinant hyperthermoacidophilicα-amylase PFA from Pyrococcus furiosus in Bacillus subtilis WB600.The signal peptide YfkN achieved the highest extracellularα-amylase activity.Then we used saturation mutagenesis of Ile3 and Gln4 of the signal peptide YfkN as a novel approach to improve the secretion of PFA.The culture supernatant of the recombinant strain contained the signal peptide I3G/Q4R and had remarkableα-amylase activity(up to 715 U/mL).The purifiedα-amylase PFA showed maximal activity at pH 5.0 and 100℃,and its half-life at 100℃was about 13 h with or without 5 mmol/L Ca2+added.To the best of our knowledge,we obtained higher yield of PFA in a food-grade host,which may benefit the industrial production and application of PFA.