Zhuoning Cao,Chengqiang Li,Hong Jiang,Francesco Secundo,Xiangzhao Mao
β- N -acetylhexosaminidases garnered attention in the enzymatic synthesis of lacto- N -triose II (LNT2) as the backbone precursor of human milk oligosaccharides (HMOs). In this study, β- N -acetylglucosaminidases Hex(Sv)-2557 from Streptomyces violascens ATCC 27968 was engineered based on a stabilizing intermediate strategy to improve its transglycosylation activity for LNT2 synthesis. A mutant Hex(Sv)-2557(D297K) with a transglycosylation activity of 38.4 U/mg with p NP-GlcNAc −1.9-fold higher than that of Hex(Sv)-2557– was obtained and characterized. Instead, the hydrolase activity of the mutant was 73 % lower compared to the wild-type enzyme. Importantly, the mutant can use N -acetyl chitobiose (GlcNAc 2 ) as the donor for LNT2 synthesis. The LNT2 yield of 14.85 % was obtained when the synthetic reaction, catalyzed by the mutant Hex(Sv)-2557(D297K), started from whey powder and GlcNAc 2 -prepared from chitin by chitinase ChiA and ChiB. This study has altered the donor for the action by directed modification and promoting the high-value utilization of whey powder and chitin.
Chengqiang Li,Zhuoning Cao,Hong Jiang,Francesco Secundo,Xiangzhao Mao
β-N-Acetylhexosaminidases have attracted much attention in the enzymatic synthesis of lacto-N-triose II (LNT2) as a backbone precursor of human milk oligosaccharides (HMOs). In this study, a novel glycoside hydrolase (GH) 20 family β-N-acetylhexosaminidase, FlaNag2353, from Flavobacterium algicola was biochemically characterized and applied to synthesize LNT2. FlaNag2353 displayed optimal activity to p-nitrophenyl N-acetyl-β-d-glucosaminide (pNP-GlcNAc) at 40 °C and pH 8.0. In addition to its excellent hydrolysis activity toward pNP-GlcNAc and chitooligosaccharides, FlaNag2353 showed trans-glycosylation activity. Under conditions of pH 9.0 and 55 °C for 2 h and utilizing 200 mM lactose and 10 mM pNP-GlcNAc, FlaNag2353 synthesized LNT2 with a conversion ratio of 4.15% calculated from pNP-GlcNAc. Moreover, when applied to LNT2 synthesis with 10 mM pNP-GlcNAc and 9.7% (w/v) industrial waste whey powder, FlaNag2353 achieved a conversion ratio of 2.39%. This study has significant implications for broadening the applications of GH20 β-N-acetylhexosaminidases and promoting the high-value utilization of whey powder.
Human milk oligosaccharides (HMOs) are significant constituents of breast milk and have potential anti-inflammatory bioactivities. We compared the immunomodulatory effects of lacto-N-biose I (LNB), lacto-N-triose II (LNT II), galacto-oligosaccharides (GOS), and their combinations (GOS+LNB, GOS+LNT II, LNB+LNT II, m/m=1:1) in lipopolysaccharide-induced RAW264.7 macrophages. LNT II and LNB+LNT II efficiently inhibited inflammation-related gene expression and enhanced phagocytosis and anti-inflammatory ability, which may be attributed to their N-acetylglucosamine structure. Three milk powder prototypes (control, LNT II, and LNB+LNT II) were formulated and characterized, exhibiting favorable physicochemical properties. In-vitro simulated digestion coupled with caucasian colon adenocarcinoma (Caco-2) inflammation model demonstrated that the milk powder digestives inhibited tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) release while promoting interleukin-10 (IL-10) production. The digestives of the three milk powders could also inhibit the decrease in transmembrane electrical resistance and the increase in intestinal permeability, playing a crucial role in maintaining intestinal homeostasis. This study will contribute to the functional evaluation and product development of HMOs and inform milk powder design.
危险品化学品经营许可证（带存储）