甘油糖脂由糖与甘油脂通过糖苷键连接而成,具有抗肿瘤、抗病毒和抗炎等多种生物活性,已成为当前研究的热点。目前获得甘油糖脂的方法包括植物提取法及化学合成法。植物提取法提取率及纯度较低;化学合成法对反应条件和分离设备要求高,产品不易分离,产生的废液污染环境。该研究采用基因工程方法构建海洋杆菌Candidatus pelagibacter sp.HTCC7211中的糖基转移酶Agt高活性突变体重组质粒,实现在大肠杆菌E.coli BL21-CodonPlus (DE3)-RIL中的表达,进一步研究其酶活性,优化催化合成单葡萄糖甘油二酯的最佳反应条件及其提取方法。结果表明:构建的突变体T257S可将二磷酸尿苷-葡萄糖(uridine diphosphate gluscose,UDP-Glu)和甘油二酯高效地转化为单葡萄糖甘油二酯,其催化效率是野生型合成酶Agt的1.56倍;采用环己烷/乙酸乙酯法提取单葡萄糖甘油二酯,最高提取率达84.7%;在最佳反应条件酶浓度8 U/mL、UDP-Glu质量浓度500 mg/L、温度35 ℃和pH 8.5条件下,转化率和纯度分别达68.5%和93.8%。因此利用突变体T257S制备单葡萄糖甘油二酯具有转化率高、产物纯度高和合成步骤较少等优势,具有良好的工业应用前景。
Glycerolglycolipid is composed of sugar and glyceride linked by glycosidic bond. It has many biological activities, such as anti-tumor, anti-virus and anti-inflammatory, and has become the focus of current research. The traditional production method of glycerolglycolipid is extracted from plant, which has low extraction rate and purity. The chemical synthesis method requires high reaction conditions and separation equipment, the product is not easy to separate, and the waste liquid pollutes the environment. In this study, the recombinant mutant plasmids of glycosyltransferase Agt from marine bacterium Candidatus pelagibacter sp. HTCC7211 were constructed by genetic engineering method to realize the expression in Escherichia coli BL21-CodonPlus (DE3)-RIL. The enzyme activity, the optimum reaction conditions and extraction method of catalytic synthesis of monoglucosyl diacylglycerol were further researched. The results showed that the mutant T257S could efficiently convert UDP-glucose and diacylglycerol into monoglucosyl diacylglycerol, and catalytic efficiency was 1.56 times higher than that of wild-type enzyme. Monoglucosyl diacylglycerol was extracted by cyclohexane/ethyl acetate method, and the extraction rate was 84.7%. Under the optimum reaction conditions (enzyme 8 U/ml, UDP-glucose 500 mg/L, 35 ℃ and pH 8.5), the conversion rate and purity could reach 68.5% and 93.8%, receptively. Therefore, the preparation of monoglucosyl diacylglycerol by mutant T257S has the advantages of high conversion, high product purity and less synthetic steps, which has a good industrial application prospect.
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