Identification and Characterization of a Novel α-L-Fucosidase from Enterococcus gallinarum and Its Application for Production of 2′-Fucosyllactose

Abstract

2′-fucosyllactose (2′FL) is an important nutrient in human milk that stimulates beneficial microbiota and prevents infection. α-L-fucosidase is a promising component for 2′FL synthesis. In this study, a soil-oriented α-L-fucosidase-producing strain from Enterococcus gallinarum ZS1 was isolated. Escherichia coli was employed as a host for cloning and expressing the α-L-fucosidase gene (entfuc). The EntFuc was predicted as a member of the GH29 family with a molecular mass of 58 kDa. The optimal pH and temperature for the activity of EntFuc were pH 7.0 and 30 °C, respectively. The enzyme exhibited a strictly specific activity for 4-Nitrophenyl-α-L-fucopyranoside (pNP-Fuc) and had a negligible effect on hydrolyzing 2′FL. EntFuc could catalyze the synthesis of 2′FL via transfucosylation action from pNP-Fuc and lactose. The yield of 2′FL reached 35% under optimal conditions. This study indicated that EntFuc with a high conversion rate is a promising enzyme source for the biosynthesis of 2′FL.

α-L-Fucosidases from an Alpaca Faeces Metagenome: Characterisation of Hydrolytic and Transfucosylation Potential

“…To summarise, α-L-fucosidases investigated were stable at pH 6.0-9.0. The previously described enzymes of the GH29 family are stable in the pH range from 4 to 11: Afc2 at pH 5-11 [40], EntFuc at pH 5-8 [38], and BT_2970 and BT_2192 at pH 4-9 [28].…”

“…The marine fucosidases FucWf1, FucWf2, and FucWf3 were most active at pH 6.3 [37]. The activities of soil fucosidases EntFuc, Mfuc1-5, and Mfuc7 were the highest at pHs 6 or 7 [24,38], while the fucosidase Mfuc6 was the most active at pH 9 [24]. The highest activity in an alkaline environment (pH 9) was determined for fucosidase rTfFuc1 from the periodontal pathogen Tannerella forsythia [39].…”

“…The relative activity of Fuc25C was higher than 75% at 15 • C and 25 • C. Lower temperatures are more specific for marine fucosidases, e.g., FucWf1, FucWf2, and FucWf3 from Wenyingzhuangia fucanilytica [37]. A soil-oriented fucosidase EntFuc isolated from Enterococcus gallinarum showed the highest hydrolytic activity at 30 • C [38]. Fucosidases Fuc25A and Fuc25C were completely inactive at 55 • C and 45 • C, respectively, while Fuc25D and Fuc25E were at 50 • C. It can be summarised that fucosidases Fuc25A, Fuc25D, and Fuc25E probably originate from intestinal bacteria, as their peak activity temperature is close to the rectal temperature of the alpaca, 38.5-39.5 • C [43].…”

“…Lower temperatures are more specific for marine fucosidases, e.g., FucWf1, FucWf2, and FucWf3 from Wenyingzhuangia fucanilytica [37]. A soil-oriented fucosidase EntFuc isolated from Enterococcus gallinarum showed the highest hydrolytic activity at 30 °C [38]. Fucosidases Fuc25A and Fuc25C were completely inactive at 55 °C and 45 °C, respectively, while Fuc25D and Fuc25E were at 50 °C.…”

Efficient production of 2′-fucosyllactose from fructose through metabolically engineered recombinant Escherichia coli

“…HMOs have been confirmed to regulating the intestinal flora, modulate the immune system, and developing the nervous system [4][5][6][7]. The biosynthesis of HMOs has become a research hotspot due to its wide application in pharmaceutical and additives [8][9][10][11][12]. 2′-Fucosyllactose (2′-FL), the most abundant component of HMOs, has been approved by the United States Food and Drug Administration (FDA) as Generally Recognized as Safe (GRAS) [13].…”