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

doi:10.7506/spkx1002-6630-20210207-125

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

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

（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

(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/Abstract

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

中图分类号:

Q814

史然，张登娅，谷懿寰，江正强，杨绍青. 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[J]. 食品科学, 2021, 42(18): 135-142.

SHI Ran, ZHANG Dengya, GU Yihuan, JIANG Zhengqiang, YANG Shaoqing. Direct Evolution of α-L-Fucosidase from Pedobacter sp. and Its Application in the Synthesis of 2’-Fucosyllactose[J]. FOOD SCIENCE, 2021, 42(18): 135-142.

[1]	史然张登娅谷懿嬛江正强 Shaoqing Yang. 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[J]. , 0, (): 0-0.
[2]	温永平唐季清韩冬孙健刘军江正强. 魔芋甘露寡糖对酸奶免疫调节活性的影响[J]. , 0, (): 0-0.
[3]	刘学强江正强余静马俊文杨行闫巧娟. 海栖热袍菌木聚糖酶B的分子改造、高效表达及其在啤酒中的应用[J]. 食品科学, 0, (): 0-0.
[4]	曾静何础阔郭建军袁林. N末端结构模块缺失对嗜热酸性III型普鲁兰多糖水解酶TK-PUL酶学性质的影响[J]. , 0, (): 0-0.
[5]	曾静，何础阔，郭建军，袁林. N末端结构模块缺失对嗜热酸性III型普鲁兰多糖水解酶TK-PUL酶学性质的影响[J]. 食品科学, 2021, 42(10): 201-208.
[6]	李延啸甄红敏华晓晗马俊文温永平闫巧娟江正强. 产黄青霉β-甘露聚糖酶的高效表达、性质及应用[J]. , 0, (): 0-0.
[7]	甄红敏，华晓晗，马俊文，温永平，李延啸，闫巧娟，江正强. 产黄青霉β-甘露聚糖酶的高效表达、性质及应用[J]. 食品科学, 2021, 42(8): 98-105.
[8]	丰国强，徐文亮，宋平，李婉珍，陶玉贵，葛飞，朱龙宝. 重组Taxus chinensis苯丙氨酸变位酶性质表征及R-β-芳香丙氨酸合成[J]. 食品科学, 2021, 42(6): 82-87.
[9]	丰国强徐文亮宋平李婉珍葛飞陶玉贵朱龙宝. 重组Taxus chinensis 苯丙氨酸氨基变位酶性质表征及合成R-β-芳香丙氨酸[J]. 食品科学, 0, (): 0-0.
[10]	江君刘军杨绍青马帅闫巧娟江正强. 黄杆菌褐藻胶裂解酶的高效表达及其在褐藻寡糖制备中的应用[J]. , 0, (): 0-0.
[11]	江君，刘军，杨绍青，马帅，闫巧娟，江正强. 黄杆菌褐藻胶裂解酶的高效表达及其在褐藻寡糖制备中的应用[J]. 食品科学, 2021, 42(4): 145-152.
[12]	张慧霞王如福李建波王争争张茜. 海藻酸钠-微孔淀粉固定化酯化酶工艺及其催陈新醋效果[J]. 食品科学, 0, (): 0-0.
[13]	张慧霞，王如福，李建波，王争争，张茜. 海藻酸钠-微孔淀粉固定化酯化酶工艺及其催陈新醋效果[J]. 食品科学, 2020, 41(10): 159-165.
[14]	黄琳马杰莹王爽王楠 Rasadi Sajeewa Rajakaruna 路福平刘逸寒. Streptomyces septatus磷脂酶D在毕赤酵母中的重组表达及酶学性质分析[J]. 食品科学, 0, (): 0-0.
[15]	黄琳，马杰莹，王爽，王楠，MORADANA GAMAGE Rasadi Sajeewa Rajakaruna，路福平，刘逸寒. Streptomyces septatus磷脂酶D在毕赤酵母中的重组表达及酶学性质分析[J]. 食品科学, 2020, 41(8): 100-107.

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

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

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价