Human milk oligosaccharides (HMOs) represent the third most abundant components in human milk, following lactose and lipids, accounting for approximately 8 % of the total breast milk content (Kobata, 2010). Comprising over 200 types of non-digestible, non-nutritive carbohydrates, the majority of HMOs exist in free form in human milk. 2’-FL is the most predominant HMO, constituting approximately 31 % of the total HMOs (Anderson and Donald, 1981, Sprenger et al., 2017, Thum et al., 2021). 2’-FL exhibits multifunctional bioactivities, including modulation of gut microbiota, inhibition of pathogen adhesion, immune regulation, and promotion of neural system development and repair (Vandenplas et al., 2018). Approved as a food ingredient in the United States and the European Union, 2’-FL is incorporated into infant formula, dietary supplements, and medical foods (Bych et al., 2018). Its exceptional physiological properties position it as a critical ingredient for humanizing infant formula, driving significant market potential and necessitating industrial-scale production. Historically, 2’-FL was isolated and purified directly from human milk, but limited availability, extraction challenges, and high costs of raw materials render this approach impractical for industrial demands (Zhou et al., 2021). Enzymatic synthesis has become the primary method for industrial production of 2’-FL due to its advantages of environmental safety and high selectivity.

α-L-Fucosidase is a neutral exo-glycoside hydrolase that catalyzes the hydrolysis of fucosidic bonds to release L-fucose residues from oligosaccharides and glycoconjugates (Zeuner et al., 2014). This enzyme facilitates the cleavage of macromolecular carbohydrates in infants, enhancing intestinal absorption of fucose from breast milk, thereby contributing to the maturation of their immune system. Under specific conditions, it can also mediate transglycosylation reactions to synthesize fucosidic linkages, such as the enzymatic synthesis of 2’-FL from pNP-Fuc and D-lactose. Notably, α-L-fucosidase exhibits significant application potential in 2’-FL synthesis, biopharmaceutical production, and tumor biomarker detection. α-L-fucosidases are widely distributed with diverse characteristics; those suitable for 2’-FL synthesis are predominantly derived from microbial sources, particularly bacterial genera such as Pedobacter sp. (Shi et al., 2020), Paenibacillus sp. (Thøgersen et al., 2019), and Thermotoga sp. (Guzmán-Rodríguez et al., 2018a), while fungal sources (e.g., Fusarium graminearum) are relatively limited (Zeuner et al., 2018). The α-L-fucosidase identified by Lezyk et al. in soil metagenomes can synthesize 2’-FL from pNP-Fuc and lactose with a yield of approximately 6 % (Lezyk et al., 2016). The F. graminearum α-L-fucosidase synthesizes 2’-FL using xyloglucan and lactose as substrates, achieving a yield of about 14 % (Rodriguez-Diaz et al., 2011). The Thermotoga maritima α-L-fucosidase catalyzes the synthesis of 2’-FL from pNP-Fuc and lactose with a yield of approximately 32.5 % (Guzmán-Rodríguez et al., 2018b). Recently, α-L-fucosidase from Pedobacter sp. CAU209 was reported with poor thermal stability hindering its application in industrial-scale 2’-FL production (Shi et al., 2023). Therefore, prospecting thermostable natural α-L-fucosidases or engineering existing ones to enhance thermal stability is crucial for advancing their application in 2’-FL synthesis.

This study aims to enhance the thermal stability of α-L-fucosidase (EC 3.2.1.51) derived from Pedobacter sp. CAU209 (PbFuc) through a combined strategy of consensus-guided engineering and directed evolution. Potential amino acid mutation sites influencing PbFuc thermostability were predicted through multiple sequence alignment, and thermostability-enhanced mutants were subsequently obtained via high-throughput screening of mutant libraries. Beneficial mutation sites were integrated to generate the combinatorial mutant (M6), which exhibited significantly improved thermostability compared to wild-type PbFuc (WT). Finally, the mechanism underlying the effects of amino acid substitutions on PbFuc thermostability was analyzed using circular dichroism (CD), fluorescence spectroscopy, and molecular dynamics (MD) simulations. This study contributes to promoting the application of α-L-fucosidase from Pedobacter sp. in the industrial production of 2’-FL.