Chitin

Enrichment medium: Chitin 10 g/L, (NH₄)₂SO₄ 10 g/L, NaCl 0.5 g/L, K₂HPO₄ 0.7 g/L, KH₂PO₄ 0.3 g/L, MgSO₄ 0.5 g/L, pH 7.0.
Selective medium: colloidal Chitin 10 g/L, (NH₄)₂SO₄ 10 g/L, NaCl 0.5 g/L, K₂HPO₄ 0.7 g/L, MgSO₄ 0.5 g/L, agar 20 g/L, pH 7.20.
Differential medium: colloidal Chitin 10 g/L, (NH₄)₂SO₄ 10 g/L, NaCl 0.5 g/L, K₂HPO₄ 0.7 g/L, MgSO₄ 0.5 g/L, p-Nitroacetanilide 0.2 g/L, agar 20 g/L, pH 7.20.
Luria Bertani (LB) medium: tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, pH 7.50.
Fermentation medium: glucose 40 g/L, peptone 20 g/L, NaCl 0.5 g/L, K₂HPO₄ 0.7 g/L, KH₂PO₄ 0.3 g/L, CaCl₂ 0.5 g/L, MgSO₄ 0.5 g/L, pH 7.20.
All media were autoclaved at 121°C for 20 min before used. Chitin was purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). Colloidal Chitin was prepared as described previously (Pareek et al., 2013a (link)). Briefly, 10 g Chitin was completely dissolved in 200 mL HCl (32%, v/v) at room temperature. The solution was filtered with glass wool in pre-cooled distilled water until the dense white precipitate (colloidal Chitin) was formed. After centrifuging at 10,000 rpm and 4°C for 10 min, the collected colloidal Chitin was washed several times with distilled water until the pH reached neutrality, and then freeze dried.

The assay was performed as described by Tian et al. (2021) (link), with some modifications. In brief, 500 µL of protein solution containing 30 μg/mL E. coli-produced BdLM1 protein was incubated with 5 mg chitin, chitosan, cellulose, or xylan (Yuanye, Shanghai, China) in a 100 rpm shaker at 4°C for 6 h. The samples were centrifuged at 13,000 g for 5 min. The supernatants were collected and concentrated to a volume of approximately 100 µL. The pellets were washed three times with incubation buffer, and then resuspended in 100 µL demineralized water. Then, 50 µL of the pellet solution or the supernatant were individually incubated with 50 µL of SDS-PAGE protein loading buffer (2×; 200 mM Tris-HCl, pH 6.5, 0.4 M dithiothreitol, 8% sodium dodecyl sulfate, 6 mM bromophenol blue, and 40% glycerol) at 95°C for 10 min. Samples were analyzed with a Western blot using anti-His antibodies. Photos were taken using Azure Biosystems (Azure, Dublin, CA, USA) in a custom setting.

Chitin was obtained from Shanghai Yuanye Bio-technology Co., Ltd. (Shanghai, China), while Fuc was purchased from Saien Biological Technology Co., Ltd. (Chengdu, Sichuan, China). Sunflower seed oil was obtained from Beijing Jinshicang Grain and Oil Trading Co., Ltd. (Beijing, China). Nile red, calcofluor white stain dyes, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were obtained from Sigma Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade or higher (Beijing Chemical Reagent Co., Beijing, China).