PDF(Pubmed)
Abstract：
Alpha-amylases are crucial hydrolase enzymes which have been widely used in food, feed, fermentation, and pharmaceutical industries. Methods for low-cost production of α-amylases are highly desirable. Soybean seed, functioning as a bioreactor, offers an excellent platform for the mass production of recombinant proteins for its ability to synthesize substantial quantities of proteins. In this study, we generated and characterized transgenic soybeans expressing the α-amylase AmyS from Bacillus stearothermophilus. The α-amylase expression cassettes were constructed for seed specific expression by utilizing the promoters of three different soybean storage peptides and transformed into soybean via Agrobacterium-mediated transformation. The event with the highest amylase activity reached 601 U/mg of seed flour (one unit is defined as the amount of enzyme that generates 1 micromole reducing ends per min from starch at 65 °C in pH 5.5 sodium acetate buffer). The optimum pH, optimum temperature, and the enzymatic kinetics of the soybean expressed enzyme are similar to that of the E. coli expressed enzyme. However, the soybean expressed α-amylase is glycosylated, exhibiting enhanced thermostability and storage stability. Soybean AmyS retains over 80% activity after 100 min at 75 °C, and the transgenic seeds exhibit no significant activity loss after one year of storage at room temperature. The accumulated AmyS in the transgenic seeds represents approximately 15% of the total seed protein, or about 4% of the dry seed weight. The specific activity of the transgenic soybean seed flour is comparable to many commercial α-amylase enzyme products in current markets, suggesting that the soybean flour may be directly used for various applications without the need for extraction and purification.
摘要：
α-淀粉酶是重要的水解酶,在食品中得到了广泛的应用。饲料，发酵，和制药行业。低成本生产α-淀粉酶的方法是非常理想的。大豆种子,作为一个生物反应器，为大量生产重组蛋白提供了一个极好的平台，因为它能够合成大量的蛋白质。在这项研究中,我们从嗜热脂肪芽孢杆菌中产生并表征了表达α-淀粉酶AmyS的转基因大豆。利用三种不同大豆贮藏肽的启动子构建α-淀粉酶表达盒，用于种子特异性表达，并通过农杆菌介导的转化转化转化到大豆中。具有最高淀粉酶活性的事件达到601U/mg种子粉(一个单位定义为在65°C下在pH5.5乙酸钠缓冲液中从淀粉每分钟产生1微摩尔还原末端的酶的量)。最佳pH值,最佳温度,大豆表达酶的酶动力学与大肠杆菌表达酶相似。然而,大豆表达的α-淀粉酶是糖基化的，表现出增强的热稳定性和储存稳定性。大豆AmyS在75°C下100分钟后保持超过80％的活性，在室温下储存一年后，转基因种子没有明显的活性损失。转基因种子中积累的AmyS约占总种子蛋白的15％。或种子干重的约4%。转基因大豆种子粉的比活性与目前市场上许多市售的α-淀粉酶产品相当，表明大豆粉可以直接用于各种应用，而无需提取和纯化。
