摘  要

酶催化转化芦丁，通过对天然产物的糖基定向改造，可显著提高异槲皮苷生产速率和选择性。然而，天然来源的糖苷酶含量低，分离纯化步骤繁琐，限制了工业化应用。因此，本文以定向水解芦丁生物转化合成异槲皮苷为目标，用基因工程手段构建纯酶生产菌种，用以制备纯α-L-鼠李糖苷酶 (RhaA)，并用荧光蛋白作为标记物来研究糖苷酶水解工艺。主要研究结果如下：

(1) 将α-L-鼠李糖苷酶基因(rhaA)和荧光蛋白cDNA的融合表达载体，并将其导入大肠杆菌Escherichia coli BL21(DE3)中，构建BL21-pET21a-rhaB1-EGFP表达菌株，并进行双酶切验证重组质粒。

(2) 诱导重组菌株进行融合表达，筛选最佳诱导温度、诱导时间和诱导物浓度。结果表明，诱导温度选为16 ℃，诱导时间范围选为16 h，诱导物浓度选为400 μM。

(3) 用镍柱对目的蛋白进行纯化，利用SDS-PAGE法、高效液相色谱法、Western Blot法等方法对其表征，测定粗酶液和纯酶液的酶活。结果表明，与RhaB1粗酶液的活性相比，纯化RhaB1的相对活性为62.3±2.2%，酶活力损失了37.7%。

(4) 对目的蛋白催化转化芦丁合成异槲皮苷过程进行产物结构验证、动力学表征和立体选择性分析。结果表明，RhaB1粗酶液水解pNPR的Km值和最大反应速率rmax分别为2.175 g/L和0.355 [g/(L×min)]，rmax/Km=0.15 min-1。在反应温度35 °C、pH 5.0-6.0、反应时间8-10 h时，RhaB1粗酶液水解芦丁合成异槲皮苷的得率为98.3 ± 3.8%。

关键词：鼠李糖苷酶；绿色荧光蛋白；融合表达载体构建；芦丁；异槲皮苷

Abstract

Biocatalysis has become an efficient approach to sugar-based directional transformation of rare natural products, which could significantly increase the reaction rate and selectivity of glycosidase. However, the natural glycosidase with low content was tedious to purify, which limited the application in industrialization. Therefore, in the present study, the isoquercitrin production from rutin by the directional biotransformation was developed. The recombinant strain was cultured to produce α-L-rhamnosidase by means of genetic engineering, and to study the glycosidase hydrolysis using green fluorescent protein as a marker. The main research contents are as follows:

(1) The cDNA of α-L-rhamnoside and green fluorescent protein were introduced into the E.coli plasmid to construct BL21-pET21a-rhaB1-EGFP expression strain. The recombinant plasmid was confirmed by restriction enzyme digestion.

(2) The fusion recombinant strain was induced to expressed, and single factor experiments were conducted to select the optimum induction temperature, induction time and inducer concentration. The experimental results showed that the optimum induction conditions were 16 °C, 16 h, and 400 μM, respectively.

(3) The target protein was purified by nickel column. The characterization and enzymatic property of the fusion protein were determined by SDS-PAGE, high performance liquid chromatography and Western Blot, and the activities of crude and purified enzymes were determined. The results showed that, comparing with the crude RhaB1, the activity of purified RhaB1 was decreased by 37.7 %.

(4) The product structure verification, kinetic analysis and stereoselectivity analysis were conducted for the conversion of rutin to isoquercition by the enzyme prepared. The results indicated that, with pNPR as substrate, the Km value and maximum reaction rate of hydrolysis of RhaB1 crude enzyme solution were 2.175 g/L and 0.355 [g/ (L * min), respectively (rmax/Km=0.15, min-1). When the reaction temperature was 35 °C, pH was 5.0-6.0, the reaction time was 8-10 h, the yield of isoquercetin was 98.3±3.8 %.

Keyword: Rhamnosidase; fluorescent protein; fusion expression vector; rutin; isoquercitrin

毕业设计（论文）题目：

鼠李糖苷酶与绿色荧光蛋白融合表达载体的构建及其催化特性