食品科学 ›› 2021, Vol. 42 ›› Issue (18): 135-142.doi: 10.7506/spkx1002-6630-20210207-125

• 生物工程 • 上一篇    下一篇

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

史然,张登娅,谷懿寰,江正强,杨绍青   

  1. (中国轻工食品生物工程重点实验室,中国农业大学食品科学与营养工程学院,北京 100083)
  • 发布日期:2021-09-29
  • 基金资助:
    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)

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

SHI Ran, ZHANG Dengya, GU Yihuan, JIANG Zhengqiang, YANG Shaoqing   

  1. (Key Laboratory of Food Bioengineering (China National Light Industry), College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China)
  • Published:2021-09-29

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。

关键词: α-L-岩藻糖苷酶;定向进化;底物特异性;转糖苷活性;2’-岩藻糖基乳糖

Abstract: 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.

Key words: α-L-fucosidase; direct evolution; substrate specificity; transglycosylation activity; 2’-fucosyllactose

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