Xylose

Cellobiose, sorbose, xylose, mannose, glucuronic acid, ribose, trehalose, and fucose were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); arabinose and raffinose were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Shanghai, China); and fructooligosaccharides were purchased from Bao Lingbao Biotechnology Co., Ltd. (Dezhou, Shandong Province, China).

Corncob from Inner Mongolia, China, were placed in sealed plastic bags to dry naturally for about three months, ground to powder, and screened through 80 mesh before being used. The weight loss of corncob powder was less than 0.01 g/g in three months. The main components of the corncob were determined by the NREL LAP method, resulting in 35% cellulose content, 34% hemicellulose content, and 19% lignin content.
Anhydrous zinc chloride, concentrated hydrochloric acid (12 mol/L), aqueous ammonia (25–28 w%), ethyl acetate, choline chloride, oxalate dihydrate, 1,4-butanediol, glucose, xylose, furfural, 5-HMF were all purchased from Sinopharm Chemical Reagents Co., LTD. (Shanghai, China). Microcrystalline cellulose, purchased from Shanghai Aladdin reagent company (Shanghai, China). Ammonia gas, analytical pure, purchased from Anhui Hefei Hengxing Industrial Gas Co., Ltd. (Hefei, China), stored in cylinders. The flow rate is 1 mL/s.
Chromatographic grade acetonitrile for HPLC was purchased from Merck (Rahway, NJ, USA). Ultrapure water was prepared via a Milli-Q system (18.2 MΩ, Millipore, Burlington, MA, USA). The hydrochloric acid (2 mol/L) used in the experiment was obtained by diluting the concentrated hydrochloric acid and ultrapure water.

The strains and plasmids used in this study are listed in Table 2. B. licheniformis ATCC 9945a was obtained from the Culture and Information Center of Industrial Microorganisms of China Universities (Wuxi, China) and used for transcriptional studies. B. licheniformis GM2, a B. licheniformis ATCC 9945a derivative that lacks amylase (encoded by amyL) and protease (encoded by aprE), served as an expression host.
Fifteen milliliters of LBG medium (lysogenic broth with 10 g/L glucose from Sinopharm Chemical, Shanghai, China) was used to activate a seed culture stored in a glycerol stock. Cultivation was performed in a 50 mL Erlenmeyer flask at 37 °C with shaking on a XT 5518 rotary shaker (Xutemp, Hangzhou, China) at 250 rpm. Fermentation medium (containing 20 g/L peptone, 10 g/L yeast extract, 5 g/L corn steep powder, 9.12 g/L K₂HPO₄, 1.36 g/L K₂HPO₄, 0.5 g/L CaCl₂, and 0.5 g/L MgSO₄·7H₂O) was inoculated with 3% (w/v) of the activated seed culture, and then 30 mL of the medium was transferred to a 250 mL Erlenmeyer flask and cultivated at 37 °C with shaking on a rotary shaker at 250 rpm. Glucose, xylose (Sinopharm Chemical, Shanghai, China), and corn steep powder (Sinopharm Chemical, Shanghai, China) were autoclaved separately and then aseptically added to the medium as a concentrated solution.

Thin-layer chromatography (TLC) was firstly applied to detect the hydrolysis products of S7-xyl and the mutant 254RL1 on xylan. The reaction mixtures were spotted on TLC Silica gel 60F_{10–20 cm} (EMD/Merck, Darmstadt, Germany). and developed with a mixture of n-butanol, acetic acid and water (3:3:1, v/v/v). Spots were stained using 5% sulfuric acid and 95% phosphoric acid. Xylose (from Sinopharm Group, China), xylobiose, xylotriose, and xylotetraose (from Megazyme, Ireland) were used as standards.

Pyropia yezoensis was obtained from Seaweed Research Center, National Institute of Fisheries Science (Mokpo, South Korea). The triglycerides (TG) assay kit and total protein (TP) assay kit were provided by Shenzhen Icubio Biomedical Technology Co., Ltd. (Shenzhen, China). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). TransScript All-in-One First-Strand cDNA Synthesis Supermix for qPCR, and TransStart Top Green qPCR SuperMix were purchased from TransGen Biotech (Beijing, China). Mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Thiazolyl blue tetrazolium bromide (MTT) and dimethyl sulfoxide were purchased from BBI Life Sciences (Shanghai, China). Minimum essential medium (MEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NE, USA). Penicillin-streptomycin solution (100×) was obtained from Biosharp Life Sciences (Anhui, China). Dextran standards were purchased from China Pharmaceutical Biological Products Analysis Institute (Beijing, China). Palmitic acid (PA) was provided by Kunchuang Biotechnology (Xi’an, China). All other chemical reagents used were of analytical grade.

E. coli DH5α and the pUC18 vector were used for gene cloning. E. coli BL21 (DE3) was used as a host for α-L-fucosidase gene expression. pET-28a (+) was used as the expression vector. A restriction endonuclease kit was obtained from TaKaRa (Tokyo, Japan). 4-Nitrophenyl-α-L-fucopyranoside (pNP-Fuc), 4-Nitrophenyl-α-D-galactopyranoside (pNP-α-Gal), 4-Nitrophenyl-β-D-galactopyranoside (pNP-β-Gal), 4-Nitrophenyl-α-D-glucosaminide (pNP-α-Glu), 4-Nitrophenyl-β-D-glucosaminide (pNP-β-Glu) and pNP-α-D-mannopyranoside were obtained from Sigma Chemical Company (St. Louis, MO, USA). 2′FL was supplied by Sangon Biotech (Shanghai, China). Lactose, glucose, gaLactose, xylose, fructose, and sucrose were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).