交替糖作为一种新型功能性低聚糖具有良好的益生作用。交替糖在自然界中的含量较少,主要通过发酵法和酶法制备,但目前交替糖蔗糖酶(alternansucrase,ASR)活性较低成为制约交替糖应用的主要因素。为促进交替糖的应用,该研究合成了密码子优化后的交替糖蔗糖酶基因,将其首次在毕赤酵母GS115中成功重组表达。3 L发酵罐高密度发酵72 h结果显示,重组菌发酵上清液中ASR酶活力为34.5 U/mL,约是摇瓶水平的12倍,也是目前重组ASR的最高表达水平。酶学性质研究表明,重组ASR的最适pH和最适温度分别是pH 6.0和55 ℃,Km和kcat分别是31.97 mmol/L,0.36 s-1。以蔗糖、蔗糖和麦芽糖等其他小分子糖为底物,在40 ℃, pH 5.5条件下利用重组ASR制备交替糖。将催化产物经薄层色谱初步分析,鉴定得出催化产物中生成了大量交替糖寡糖和交替糖多糖。该研究首次实现了交替糖蔗糖酶基因在毕赤酵母中高水平重组表达,并催化制备了交替糖,为ASR的异源表达及规模化制备交替糖提供了良好基础。
Alternan is a novel functional oligosaccharides which has good prebiotic properties. The content of alternate sugars is less in nature and mainly prepared by microbial fermentation and enzyme conversion. Currently, low activity of alternansucrase prevents its application in largescale. To enhance the enzyme expression, the alternansucrase gene was synthesized and heterologously expressed in Pichia pastoris GS115 for the first time. After 72 h high-density fermentation in 3 L fermenter, the enzyme activities in supernatant reached 34.5 U/ml, which was about 12 times than that of shake flask fermentation, and reached the highest level of recombinant alternansucrase at present. The optimum pH and temperature of recombinant ASR were 6.0 and 55℃, Km and kcat were 31.97 mmol/L and 0.36 s-1, respectively. In addition, alternan was prepared by using sucrose or sucrose and other receptors as substrates at 40°C and pH 5.5. It was identified by TLC that both oligoalternan and polyalternan were produced in the catalytic products. The alteransucrase gene was successfully expressed in P. pastoris for the first time, which will provide a basis for the heterologous expression of alteransucrase and the large-scale preparation of alternan.
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