地杆菌α-L-岩藻糖苷酶的分子改造及其在合成2’-岩藻糖基乳糖中的应用

对地杆菌α-L-岩藻糖苷酶进行分子改造，以提高其酶法合成2’-岩藻糖基乳糖（2’-fucosyllactose，2’-FL）的转化效率。利用易错聚合酶链式反应构建了α-L-岩藻糖苷酶的突变体文库，筛选得到一个合成2’-FL转化率提高的突变酶（mPbFuc29A1）。酶学性质研究结果表明mPbFuc29A1的最适pH值和温度分别为pH 5.0和40 ℃，最适温度较野生型PbFuc29A1提高了5 ℃；突变酶水解2’-FL的比活力提高到3 倍，但是水解4-硝基苯基-α-L-岩藻糖苷（4-nitrophenyl-α-L-fucopyranoside，pNP-FUC）和3’-岩藻糖基乳糖（3’-fucosyllactose，3’-FL）的比活力分别降低了22.8%和52.5%。以pNP-FUC和乳糖为底物，采用mPbFuc29A1酶法催化合成2’-FL和3’-FL，转化率分别为23.6%和56.4%，其中2’-FL的转化率较PbFuc29A1提高了9.1%。通过序列及定点突变分析发现mPbFuc29A1的氨基酸序列中有2 个位点发生突变（Asp21Val和Glu266Lys），其中位于loop区的Glu266Lys可能是mPbFuc29A1底物特异性和转糖苷产物组成发生改变的关键。优良的酶学特性使mPbFuc29A1在2’-FL合成中具有较大的应用潜力。

Direct Evolution of α-L-Fucosidase from Pedobacter sp. and Its Application in the Synthesis of 2’-Fucosyllactose

The α-L-fucosidase from Pedobacter sp. was modified by direct evolution for improved 2’-fucosyllactose (2’-FL) synthesis. A mutation library of α-L-fucosidase (PbFuc29A1) was constructed by error-prone PCR, and one mutant (mPbFuc29A1) capable of providing improved conversion ratio of 2’-FL was identified and characterized. The modified enzyme (mPbFuc29A1) showed the highest activity at pH 5.0 and 40 ℃ (compared to 35 ℃ for the wild-type PbFuc29A1), respectively. In comparison with PbFuc29A1, the specific activity of mPbFuc29A1 towards 2’-FL was increased by three folds, while that towards 4-nitrophenyl-α-L-fucopyranoside (pNP-FUC) and 3’-fucosyllactose were decreased by 22.8% and 52.5%, respectively. Using pNP-FUC and lactose as the substrates, mPbFuc29A1 catalyzed the synthesis of 2’-FL and 3’-FL with conversion ratios of 23.6% (9.1% higher than that with PbFuc29A1) and 56.4%, respectively. Sequence and mutation analysis revealed that there were two mutation sites (Asp21Val and Glu266Lys) in mPbFuc29A1, and Glu266Lys (located in the loop area) may play a key role in changing the specific activity and transglycosylation property of the mutant. The excellent properties of mPbFuc29A1 may make it a great candidate in industrial synthesis of 2’-FL.