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首页> 外文期刊>Microbial Cell Factories >Production of trehalose with trehalose synthase expressed and displayed on the surface of Bacillus subtilis spores
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Production of trehalose with trehalose synthase expressed and displayed on the surface of Bacillus subtilis spores

机译：在枯草芽孢杆菌孢子表面表达和展示的海藻糖合酶生产海藻糖

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Bacillus subtilis spores have been commonly used for the surface display of various food-related or human antigens or enzymes. For successful display, the target protein needs to be fused with an anchor protein. The preferred anchored proteins are the outer-coat proteins of spores; outer-coat proteins G (CotG) and C (CotC) are commonly used. In this study, mutant trehalose synthase (V407M/K490L/R680E TreS) was displayed on the surface of B. subtilis WB800n spores using CotG and CotC individually or in combination as an anchoring protein. Western blotting, immunofluorescence, dot blot, and enzymatic-activity assays detected TreS on the spore surface. The TreS activity with CotC and CotG together as the anchor protein was greater than the sum of the enzymatic activities with CotC or CotG alone. The TreS displayed on the spore surface with CotC and CotG together as the anchoring protein showed elevated and stable specific activity. To ensure spore stability and prevent spore germination in the trehalose preparation system, two germination-specific lytic genes, sleB and cwlJ, were deleted from the B. subtilis WB800n genome. It was demonstrated that this deletion did not affect the growth and spore formation of B. subtilis WB800n but strongly inhibited germination of the spores during transformation. The conversion rate of trehalose from 300?g/L maltose by B. subtilis strain WB800n(ΔsleB, ΔcwlJ)/cotC-treS–cotG-treS was 74.1% at 12?h (350 U/[g maltose]), and its enzymatic activity was largely retained, with a conversion rate of 73% after four cycles. The spore surface display system based on food-grade B. subtilis with CotC and CotG as a combined carrier appears to be a powerful technology for TreS expression, which may be used for the biotransformation of d-maltose into d-trehalose.
机译：枯草芽孢杆菌孢子已普遍用于各种食品相关或人类抗原或酶的表面展示。为了成功展示，目标蛋白需要与锚定蛋白融合。优选的锚定蛋白是孢子的外壳蛋白。通常使用外被膜蛋白G（CotG）和C（CotC）。在这项研究中，突变型海藻糖合酶（V407M / K490L / R680E TreS）在枯草芽孢杆菌WB800n孢子的表面上分别使用CotG和CotC或结合作为锚定蛋白展示。蛋白质印迹，免疫荧光，斑点印迹和酶活性测定法在孢子表面检测到TreS。以CotC和CotG共同作为锚蛋白的TreS活性大于单独用CotC或CotG的酶促活性的总和。 Tres与CotC和CotG一起显示在孢子表面，因为锚定蛋白显示出升高的和稳定的比活性。为了确保孢子的稳定性并防止海藻糖制备系统中的孢子萌发，从枯草芽孢杆菌WB800n基因组中删除了两个萌发特异性裂解基因sleB和cwlJ。证实了该缺失不影响枯草芽孢杆菌WB800n的生长和孢子形成，但是在转化过程中强烈抑制了孢子的萌发。枯草芽孢杆菌WB800n（ΔsleB，ΔcwlJ）/ cotC-treS–cotG-treS从300？g / L麦芽糖中海藻糖的转化率在12？h（350 U / [g麦芽糖]）为74.1％。酶活性基本上得以保留，四个循环后转化率为73％。以CotC和CotG为载体的食品级枯草芽孢杆菌的孢子表面展示系统似乎是TreS表达的强大技术，可用于将d-麦芽糖生物转化为d-海藻糖。

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