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This study developed a cell-free enzymatic cascade for the sustainable production of 3,6-anhydro-D-galactose (DA) from various carrageenans, a family of sulfated polysaccharides from red algae. The bioconversion system integrated a promiscuous carrageenase (BovGH16) for initial depolymerization, two novel sulfatases (CaCgS2, exo-G4S-sulfatase; EpCgS2, exo-DA2S-sulfatase) for desulfation, and two glycoside hydrolases (CaGH127_1, β-galactosidase WcBGH) for monomerization. BovGH16 hydrolyzed κ-, ι-, and hybrid carrageenans into oligosaccharides, while CaCgS2 and EpCgS2 removed sulfate groups from galactose-4-sulfate (G4S) and 3,6-anhydro-D-galactose-2-sulfate (DA2S), respectively. CaGH127_1 and WcBGH further cleaved nonsulfated oligosaccharides to release DA and D-galactose. The system produced DA from all tested carrageenans, with the highest yield from κ-carrageenan (3.9 mM, 0.126 g/g substrate) and 2.23 mM from ι-carrageenan. Scaling up with prehydrolyzed substrates yielded 24 mM DA from 5% (w/v) κ-carrageenan hydrolysate. These studies demonstrate that the enzymatic one-pot system efficiently converts sulfated galactans to DA, a valuable sugar with anti-inflammatory and skin-whitening properties, supporting sustainable biotechnological applications.The in vitro bioconversion of carrageenans to DA was conducted through controlled enzymatic reactions. Carrageenans (κ-, ι-, hybrid types) were extracted from red algae (Kappaphycus alvarezii, Eucheuma spinosum, Chondrus crispus) and purified. Recombinant enzymes were heterologously expressed in E. coli BL21(DE3), purified via affinity chromatography, and characterized for pH/temperature optima (pH 6.5-7.0, 35-55 °C). The one-pot reaction contained 5 g/L carrageenan, BovGH16 (0.0075 mg/mL), CaCgS2 (2.5 mg/mL), EpCgS2 (0.667 mg/mL), CaGH127_1 (0.75 mg/mL), and WcBGH (0.166 mg/mL) in 20 mM Tris-HCl (pH 7.5), incubated at 37 °C for 16 hours. DA was quantified via HPLC (Rezex ROA column) and a spectrophotometric assay using DA dehydrogenase (CaDADH). Reaction intermediates were analyzed by ¹H NMR to confirm desulfation and hydrolysis efficiency.
This study developed a cell-free enzymatic cascade for the sustainable production of 3,6-anhydro-D-galactose (DA) from various carrageenans, a family of sulfated polysaccharides from red algae. The bioconversion system integrated a promiscuous carrageenase (BovGH16) for initial depolymerization, two novel sulfatases (CaCgS2, exo-G4S-sulfatase; EpCgS2, exo-DA2S-sulfatase) for desulfation, and two glycoside hydrolases (CaGH127_1, β-galactosidase WcBGH) for monomerization. BovGH16 hydrolyzed κ-, ι-, and hybrid carrageenans into oligosaccharides, while CaCgS2 and EpCgS2 removed sulfate groups from galactose-4-sulfate (G4S) and 3,6-anhydro-D-galactose-2-sulfate (DA2S), respectively. CaGH127_1 and WcBGH further cleaved nonsulfated oligosaccharides to release DA and D-galactose. The system produced DA from all tested carrageenans, with the highest yield from κ-carrageenan (3.9 mM, 0.126 g/g substrate) and 2.23 mM from ι-carrageenan. Scaling up with prehydrolyzed substrates yielded 24 mM DA from 5% (w/v) κ-carrageenan hydrolysate. These studies demonstrate that the enzymatic one-pot system efficiently converts sulfated galactans to DA, a valuable sugar with anti-inflammatory and skin-whitening properties, supporting sustainable biotechnological applications.The in vitro bioconversion of carrageenans to DA was conducted through controlled enzymatic reactions. Carrageenans (κ-, ι-, hybrid types) were extracted from red algae (Kappaphycus alvarezii, Eucheuma spinosum, Chondrus crispus) and purified. Recombinant enzymes were heterologously expressed in E. coli BL21(DE3), purified via affinity chromatography, and characterized for pH/temperature optima (pH 6.5-7.0, 35-55 °C). The one-pot reaction contained 5 g/L carrageenan, BovGH16 (0.0075 mg/mL), CaCgS2 (2.5 mg/mL), EpCgS2 (0.667 mg/mL), CaGH127_1 (0.75 mg/mL), and WcBGH (0.166 mg/mL) in 20 mM Tris-HCl (pH 7.5), incubated at 37 °C for 16 hours. DA was quantified via HPLC (Rezex ROA column) and a spectrophotometric assay using DA dehydrogenase (CaDADH). Reaction intermediates were analyzed by ¹H NMR to confirm desulfation and hydrolysis efficiency.