高效转化大豆低聚糖的果聚糖蔗糖酶的挖掘、表征及表达

Mining, characterization, and expression of a fructan sucrase for efficient conversion of soybean oligosaccharides

摘要:

大豆低聚糖因含大量蔗糖和棉子糖影响了其益生元价值。果聚糖蔗糖酶可催化蔗糖和棉子糖生成高附加值的低聚果糖和蜜二糖等组分。为获得高效转化大豆低聚糖的果聚蔗糖酶，本研究首先从我国西沙群岛及渤海湾沿海区域微生物中挖掘果聚糖蔗糖酶基因，然后对其进行酶学性质和催化性能表征，最后将其在枯草芽孢杆菌(Bacillus subtilis)中进行重组胞外表达。结果显示，从耐盐芽孢杆菌(Bacillus halotolerans)中挖掘出的新型果聚糖蔗糖酶BhLS 39，其以蔗糖和棉子糖为底物时的最适温度分别为50℃和55℃，最适pH值均为5.5，K_cat/K_m分别为3.4和6.6 L/(mmol·s)。5 U酶处理400 g/L棉子糖30 min后获得的蜜二糖转化率为84.6%，BhLS 39催化蔗糖可生成左聚型低聚果糖和果聚糖。本研究实现了BhLS 39在枯草芽孢杆菌中的重组胞外表达，并通过共表达胞内伴侣蛋白DnaK和胞外伴侣蛋白PrsA使其重组胞外活性提高到17 U/mL，是对照菌的5.2倍。本研究挖掘出的BhLS 39有利于大豆低聚糖的提质增效，也可促进其他重组蛋白在枯草芽孢杆菌中实现高效重组表达。

关键词:大豆低聚糖;左聚糖蔗糖酶;枯草芽孢杆菌;伴侣蛋白;胞外表达

Abstract:

The high content of sucrose and raffinose reduces the prebiotic value of soybean oligosaccharides. Fructan sucrases can catalyze the conversion of sucrose and raffinose to high-value products such as fructooligosaccharides and melibiose. To obtain a fructan sucrase that can efficiently convert soybean oligosaccharides, we first mined the fructan sucrase gene from microorganisms in the coastal areas of Xisha Islands and Bohai Bay and then characterized the enzymatic and catalytic properties of the enzyme. Finally, recombinant extracellular expression of this gene was carried out in Bacillus subtilis. The results showed that a novel fructan sucrase, BhLS 39, was mined from Bacillus halotolerans. With sucrose and raffinose as substrates, BhLS 39 showed the optimal temperatures of 50 ℃ and 55 ℃, optimal pH 5.5 for both, and K_cat/K_m ratio of 3.4 and 6.6 L/(mmol·s), respectively. When 400 g/L raffinose was used as the substrate, the melibiose conversion rate was 84.6% after 30 min treatment with 5 U BhLS 39. Furthermore, BhLS 39 catalyzed the conversion of sucrose to produce levan-type-fructooligosaccharide and levan. Then, the recombinant extracellular expression of BhLS 39 in B. subtilis was achieved. The co-expression of the intracellular chaperone DnaK and the extracellular chaperone PrsA increased the extracellular activity of the recombinant BhLS 39 by 5.2 folds to 17 U/mL compared with that of the control strain. BhLS 39 obtained in this study is conducive to improving the quality and economic benefits of soybean oligosaccharides. At the same time, the strategy used here to enhance the extracellular expression of BhLS 39 will also promote the efficient recombinant expression of other proteins in B. subtilis.

Key words:soybean oligosaccharides;levansucrase;Bacillus subtilis;chaperonin;extracellular expression