摘要
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。
Theα-L-fucosidase from Pedobacter sp.was modified by direct evolution for improved 2’-fucosyllactose(2’-FL)synthesis.A mutation library ofα-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.
作者
史然
张登娅
谷懿寰
江正强
杨绍青
SHI Ran;ZHANG Dengya;GU Yihuan;JIANG Zhengqiang;YANG Shaoqing(Key Laboratory of Food Bioengineering(China National Light Industry),College of Food Science and Nutritional Engineering,China Agricultural University,Beijing 100083,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2021年第18期135-142,共8页
Food Science
基金
国家自然科学基金优秀青年科学基金项目(31822037)
“十三五”国家重点研发计划重点专项(2017YFD0400200)。
