Understanding the structure and composition of recalcitrant oligosaccharides in hydrolysate using high-throughput biotin-based glycome profiling and mass spectrometry

Biomass

Corn Stover (CS) grown from Pioneer hybrid seed variety 33A14 was harvested in 2010 from Kramer farm in Wray, CO. Permission was obtained from the land owner to use this biomass for research. The samples were stored dry < 6% moisture in zip-lock bags in room temperature. The study complies with local and national guidelines. Composition analysis was performed using the NREL protocol⁴⁵. The composition was found to have 31.4% glucan, 18.7% xylan, 3.3% arabinan, 1.2% galactan, 2.2% acetyl, 14.3% lignin, 1.7% protein, and 13.4% ash.

Chemicals and enzymes

Cellic® CTec2 (138 mg protein/mL, batch number VCNI 0001) is a complex blend of cellulase ezymes, β-glucosidase and Cellic® HTec2 (157 mg protein/mL, batch number VHN00001) is a complex blend of hemicellulase enzymes were generously provided by Novozymes (Franklinton, NC, USA). Multifect Pectinase® (72 mg protein/mL) is a comlex mixture of pectine degrading enzymes was a gift from DuPont Industrial Biosciences (Palo Alto, CA, USA). The protein concentrations of the enzymes were determined by estimating the protein (and subtracting the nonprotein nitrogen contribution) using the Kjeldahl nitrogen analysis method (AOAC Method 2001.11 by Dairy One Cooperative Inc., Ithaca, NY, USA). Celite 545 was purchased from EMD Millipore (Billerica, MA). Activated charcoal (DARCO, 100 mesh particle), Avicel (PH-101), beechwood xylan and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

AFEX pretreatment

AFEX pretreatment was performed at the GLBRC (Biomass Conversion Research Laboratory, MSU, Lansing, MI, USA). Pretreatmet was carried out at 140 °C for 15 min. residence time at 60% (wt/wt) moisture with 1:1 anhydrous ammonia to biomass loading in a bench-top stainless steel batch reac-tor (Parr Instruments Company)⁴⁶. It took 30 min. for the reactor to reach 140 °C and the ammonia was rapidly released, which quickly brought the biomass to room temperature. The AFEX-pretreated corn stover (ACS) composition was similar to untreated corn stover (UT-CS).

High solids loading enzymatic hydrolysis

High solids loading 25% (w/w) (approximately to 8% glucan loading) ACSH was prepared as starting material for the large-scale production of oligosaccharides. Enzymatic hydrolysis of ACS was performed using a commercial enzymes mixture including Cellic® Ctec2 10 mg protein/g glucan (in pretreated biomass), Htec2 (Novozymes, Franklinton, NC), 5 mg protein/g glucan and Multifect Pectinase (Genencor Inc, USA), 5 mg protein/g glucan. Enzymatic hydrolysis was carried out in a 5L bioreactor with 3L working volume at pH 4.8, 50 °C, and 250 rpm. After 96 h hydrolysis, the hydrolysate was harvested by centrifugation at 6,000 rpm for 30 min and then 14,000 rpm for 30 min to remove unhydrolyzed solids. Hydrolysate was then sterile filtered through a 0.22 mm filter cup. The filtered hydrolysate was stored at 4 °C in a sterile bottle prior to charcoal fractionation.

Biomass analysis

Extractive-based compositional analyses of the biomass samples were performed according to the NREL Laboratory Analytical Procedures: Preparation of samples for compositional analysis (NREL/TP-510-42620) and determination of structural carbohydrates and lignin in biomass (NREL/TP-510-42618)⁴⁷.

Oligosaccharide analysis

Oligomeric sugar analysis was conducted on the hydrolysate liquid streams using an autoclave-based acid hydrolysis method at a 2 mL scale. Hydrolysate samples were mixed with 69.7 μL of 72% sulfuric acid in 10 mL screw-cap culture tubes and incubated in a 121 °C bench-top hot plate for 1 h, cooled on ice and filtered into High Performance Liquid Chromatography (HPLC) vials. The concentration of oligomeric sugar was determined by subtracting the monomeric sugar concentration of the non-hydrolyzed samples from the total sugar concentration of the acid hydrolyzed samples.

Analytical method

Glucose, xylose and arabinose concentrations in acid hydrolyzed biomass were analyzed using a Shimadzu HPLC system equipped with a Bio-Rad Aminex HPX-87H column equipped with automatic sampler, column heater, isocratic pump, and refractive index detector. The column was maintained at 50 °C and eluted with 5 mM H₂SO₄ in water at 0.6 mL/min. flowrate.

Liquid and solid composition analysis

The hydrolysate supernatants were diluted and analyzed for monomeric and oligomeric sugar contents. Monomeric sugars produced after enzyme hydrolysis were analyzed using an HPLC equipped with a Bio-Rad (Hercules, CA) Aminex HPX-87P column and de-ashing guard column. Column temperature was held at 80 °C and water was used as the mobile phase flowing at 0.6 mL/min. Oligomeric sugars were determined via dilute acid hydrolysis at 121 °C according to the method reported in the literature^41,48,49.

Glycome profiling

Glycome profiling of untreated, AFEX-pretreated and all unhydrolyzed biomass residues (involving preparation of sequential cell wall extracts and their mAb screenings) were carried out using the procedures previously described^27,43,50,51. To conduct glycome profiling, alcohol insoluble residue of plant cell wall materials were prepared from biomass residues and were subjected to sequential extractions with increasingly harsh reagents such as ammonium oxalate (50 mM), sodium carbonate (50 mM with sodium borodeutiride at 0.5% w/v), KOH (1 M and 4 M, both containing sodium borodeutiride at 1% w/v) and acidic chlorite as described previously^52,53. The extracts were then subjected to ELISA against a comprehensive suite of cell wall glycan-directed mAbs⁵⁰ and the mAb binding responses were represented as heat maps. Plant cell wall glycan-directed mAbs were purchased from laboratory stocks (CCRC, JIM and MAC series).

ELISA-based mAb screening of biotinylated oligosaccharides

1. 1.

   One-step biotinylation of oligosaccharides. The coupling of carbohydrates to biotin-LC-hydrazide was carried using the following procedure. Biotin-LC-hydrazide (4.6 mg/12 μmol) was dissolved in dimethyl sulfoxide (DMSO, 70 μl) by vigorous mixing and heating at 65 °C for 1 min. Glacial acetic acid (30 μl) was added, and the mixture was poured onto sodium cyanoborohydride (6.4 mg/100 μmol), which dissolved completely after heating at 65 °C for approximately 1 min. Then 5 to 8 μl of the reaction mixture was added to the dried oligosaccharides (1–100 nmol) to obtain a tenfold or greater molar excess of label over reducing ends. The reaction was carried out at 65 °C for 2 h, after which the samples were purified immediately. In labeling experiments without reduction, sodium cyanoborohydride was omitted and the samples were allowed to react at 65 °C for 2.5 h.

2. 2.

   ELISA of biotinylated oligosaccharides samples coating and wash. A 25 μL of biotinylated sample (100 μL each concentrated sample diluted in 5 mL 0.1 M Tris Buffered Saline (TBS) separately) were added to respective wells on Avidin-coated plates. The control wells were coated with 50 μl biotin at 10 μg/mL in 0.1 M TBS. DI water was used as coat for blank readings. The plates were incubated at room temperature for 2 h in the dark. The plates was washed 3 times with 0.1% skimmed milk in 0.1 M TBS using plate washing program #11 for Grenier flat 3A.

3. 3.

   Primary antibody addition and wash. A 40 μl primary antibody was added to respective wells. The microplates were incated at room temperature for 1 h in the dark. The plates were then washed 3 times with 0.1% milk in 0.1 M TBS using plate washing program #11 for Grenier flat 3A.

4. 4.

   Secondary antibody addition and wash. A 50 μl Mouse/Rat secondary antibody was aded to respective wells (dilute secondary antibody in 1:5000 proportion using 0.1% milk in 0.1 M TBS). The microplates was incubated at room temperature for 1 h in the dark. Then the microplates was washed 5 times using 0.1% milk in 0.1 M TBS using plate washing program #12 for Grenier flat 5A.

5. 5.

   Substrate Addition. A 50 μl of 3,3′,5,5′-Tetramethylbenzidine (TMB) was added to the substrate substrate (Prepare TMB substrate by adding 2 drops of buffer, 3 drops of TMB, 2 drops of hydrogen peroxide in 15 mL DI Water and vortex before use). The microplates was incubated at room temperature for 30 min. in the dark.

6. 6.

   Termination step and reading the plate. A 50 μl of 1 N sulfuric acid was added to each well and the absorbance was recorded using ELISA reader between 450 and 655 nm.

TMS sugar composition analysis

A 1 mg/mL solutions of these analytes was prepared in deionized water: arabinose, rhamnose, fucose, xylose, galacturonic acid (GalA), glucuronic acid (GlcA), mannose, glucose, galactose, N-acetylmannosamine (manNAc), N-acetyl-glucosamine (glcNAc), N-acetyl-galactosamine (galNAc), myo-inositol (internal standard). Two standards were prepared by addding the 1 mg/mL sugar solutions as Table 1. The samples were frozen at −80 °C and lyophilize until all water is removed (usually it takes about 12–18 h).

TMS sample preparation

A 100–500 μg of sample was added into a screw cap tube on analytical balance. Record the amount added. It is best to have the samples dissolved in solvent at certain concentration, added into the tube as liquid aliquots. A 20 μL of 1 mg/mL myo-inositol was used as internal standard into each sample tube. The amount of internal standard added to sample must be the same as that added into the standard tube.

Preparation of 1 M methanolic HCI (to perform up to 20 tubes)

A 8 mL of anhydrous methanol was added into a screw cap test tube. A 4 mL of 3 N methanolic HCl was then added and cap was placed and Vortex. No water used in this process.

Hydrolysis

A 500 μL 1 M methanolic HCl was added into oligosaccharideds sample and standard TMS tubes. The samples was incubate overnight (168 h) at 80 °C in heat block. Methanolysis product was dried at room temperature using the drying manifold. A 200 μL MeOH was added and dried again. The process was repeated for two times. A 200 μL methanol, 100 μL pyridine, and 100 μL acetic anhydride was added to the sample and mixed well. The sample was incubated at room temperature for 30 min. and dried. A 200 μL methanol was added and dried again.

Silylation

A 200 μL Tri-Sil was added and the capped tubes were heated for 20 min. at 80 °C and then cooled to room temperature. The samples were further dried using drying manifold to a volume of ~ 50 μL. It is important to note that we did not allow the sample to completely dry.

Gas chromatography (GC) samples preparation and injection

A 2 mL hexane was added and mix well by vortexing. Pasteur pipette tip (5–8 mm) was pack a bit of glass wool by inserting the glass wool from the top of 5–3/4 -inch pipette. The samples were centrifuged at 3000×g for 2 min. to precipitate any insoluble residue. The samples were dried until it reached 100–150 μL. About 1 µl volume was injected into the GC–MS with initial temp 80 °C and initial time 2.0 min (Table 2).

Calculation

If running standards for first time, perform TMS derivatization of each sugar separately and run them individually to procure the profiles of retention times, spectra, and distributions of peaks for each monosaccharide. These profiles will be used to identify the monosaccharide in standards mixture and samples. From the GC–MS, obtain the peak area and retention time of each sugar in standards and samples. Assign the corresponding sugar identities to the unknown peaks in samples according to the standards retention time. Because each sugar can have as many as 4 peaks, in total all the area from peaks corresponding to 1 sugar.

Calculate the detector response factor (RF) value of each sugar in the standard mixture against internal standard (in this case, inositol).

Calculate the mass of each sugar in the sample in mg using the RF value for corresponding sugar.

Calculate the mol% of each sugar Calculate the number of moles of each sugar in sample.

Calculate the total µmoles of all the sugars present in the samples. Calculate the mol% of each sugar per total moles of all sugars.