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