Glycosidic linkage analysis

Syntheses of the in vitro β-glucan polymer from both CslF6 and CesA8 were performed as described above for HPLC analysis but without added hydrolytic enzymes. After overnight synthesis at 30°C, the entire mixture was precipitated with 80% (v/v) ethanol for 5 hours at room temperature (25°C). The precipitates were collected by centrifugation at 21,200g for 20 min. The pellet were washed twice with 80% (v/v) ethanol for 10 min at 21,200g and air-dried before glycosidic linkage analysis.

Glycosidic linkages were identified and quantified as described (44), with minor modifications. Briefly, 1 mg of the freeze-dried samples was methylated in anhydrous dimethyl sulfoxide by adding 0.1 ml of methyl iodide under nitrogen and sonicating for 10 min at room temperature. This step was repeated four times to avoid undermethylation of the polysaccharides. One milliliter of dichloromethane (DCM) was subsequently added to the samples, and the methylated polysaccharides were extracted by phase partitioning against deionized water. This step was repeated three times, and the resultant combined DCM phases were evaporated under a stream of nitrogen, followed by hydrolysis at 100°C for 3 hours in 1 ml of 2 M trifluoroacetic acid under nitrogen. The hydrolysates were reduced overnight at room temperature in the presence of NaBD₄ under nitrogen and acetylated with acetic anhydride at 100°C for 12 hours. The PMAAs were recovered by evaporating the acetic anhydride solvent under a gentle stream of nitrogen and redissolving the samples in DCM. The PMAAs were purified by partitioning against deionized water, and the DCM phase was transferred to a GC vial and analyzed on an Agilent 7890B/5977B GC-MS (Agilent Technologies, Santa Clara, USA) fitted with a VF-23ms capillary column (30 m by 0.25 mm, 0.25 μm; Agilent Technologies, USA). Helium was used as carrier gas, and the oven temperature was programmed as follows: from 165° to 175°C at 1°C/min, from 175° to 195°C at 0.5°C/min, from 195° to 210°C at 2°C/min, and from 210°C to 250°C at 10°C/min, followed by a plateau at 250°C for 6.5 min (total run time of 68 min). The fragmentation patterns of the different PMAAs were interpreted by referring to the Complex Carbohydrate Research Center (CCRC) Spectral Database for PMAAs (https://glygen.ccrc.uga.edu/ccrc/specdb/ms/pmaa/pframe.html).