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

• Bioengineering • Previous Articles     Next Articles

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

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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