糖基转移酶UGT73C5在大肠杆菌中的可溶性表达研究

Soluble expression of glycosyltransferase UGT73C5 in Escherichia coli

络塞是一种抗高原反应药物的主要活性成分,可通过尿苷二磷酸糖基转移酶UGT73C5催化肉桂醇糖基化反应合成。然而UGT73C5在大肠杆菌宿主中可溶性表达极差,严重限制了其工业应用。该研究分别通过与分子伴侣共表达和与助溶蛋白标签融合表达的方式,提高UGT73C5在Escherichia coli BL21(DE3)中的可溶性表达。结果显示,除质粒pKJE7外,与分子伴侣质粒pG-KJE8、pGro7和pG-TF2共表达后均可提高UGT73C5酶活力,分别为原始酶活力(18.56 U/g细胞)的1.27、1.18、1.37倍。此外,利用硫氧还蛋白(thioredoxin,Trx)和谷胱甘肽巯基转移蛋白(glutathione S-transferase,GST)两种助溶蛋白标签,构建的融合酶Trx-UGT73C5和GST-UGT73C5的酶活力为原始酶活力的1.45、2.54倍,且纯化后的比酶活力为原始酶的80%~90%。最后,在2 mmol/L肉桂醇合成络塞的反应中,相同浓度细胞破碎液(粗酶)融合酶GST-UGT73C5比原始酶催化效率更高,4 h后转化率可以达到90%以上,最终转化率达到93.6%。因此,助溶蛋白标签融合表达法可有效提高UGT73C5在大肠杆菌中的可溶性表达,且融合酶GST-UGT73C5在络塞的酶法合成中具有更大的应用潜力。

The uridine diphosphate glycosyltransferase UGT73C5 can catalyze the glycosylation of cinnamyl alcohol for the synthesis of rosin,which is one of the main active ingredients of anti-altitude sickness drugs.However,the poor soluble expression of UGT73C5 in Escherichia coli severely limits its industrial application.Here,the soluble expression of glycosyltransferase UGT73C5 was improved by co-expression of molecular chaperone protein and fusion expression with solubilizing-tag,respectively.Results showed that besides plasmid pKJE7,pET28a-UGT73C5 co-expressed with chaperone plasmids pG-KJE8,pGro7 and pG-TF2 could increase the enzymatic activities to 1.27-,1.18-and 1.37-fold of that of UGT73C5(18.56 U/g cells).Two solubilizing protein tags,thioredoxin(Trx)and glutathione S-transferase(GST),were fused with UGT73C5.The activities of fusion enzymes Trx-UGT73C5 and GST-UGT73C5 were 1.45-and 2.54-fold of that of the UGT73C5,respectively.After purification,the specific activity of the fusion enzymes possessed 80%-90%of that of the UGT73C5.Finally,rosin synthesis from 2 mmol/L cinnamyl alcohol was performed based on crude enzymes(disruption of same concentration of cells),the fusion enzyme GST-UGT73C5 led to higher catalytic efficiency than UGT73C5.The conversion ratio of cinnamyl alcohol catalyzed by GST-UGT73C5 reached more than 90%at 4 h,and finally reached 93.6%.Therefore,the fusion expression approach is more effective for the enhanced soluble expression of UGT73C5 in E.coli,and the fusion enzyme GST-UGT73C5 is more potential in the enzymatic production of rosin.