Total carbohydrate content was determined by Bernfeld (1955) method using a xylose-based calibration curve. Xylose was determined by D-xylose assay kit (Megazyme), specifically designed to measure this sugar in samples containing up to 5 mg of glucose in the final assay reaction without any interference. Glucose was assayed enzymatically by GOPOD-FORMAT, Megazyme. Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin as the standard. Thermomyces lanuginosus xylanase, (X2753 2500 U/g) was obtained from Sigma. Thermotoga maritima endo-1,4-β-xylanase, 22 U/mg of protein (wheat arabinoxylan at pH 5.0 and 40°C) was obtained from Megazyme.
1H NMR spectra were recorded at 600.13 MHz on a Bruker DRX-600 spectrometer, equipped with a TCI Cryo Probe TM, fitted with a gradient along the Z-axis. Spectra in D2O were referenced to internal sodium 3-(trimethyl-silyl)-(2,2,3,3-2H4) propionate (Aldrich, Milwaukee, WI). 13C NMR, JMOD-1H and COSY, TOCSY, HSQC, HSQC-EDITED, HMBC (3 J: 7 and 10 Hz), NOESY (mixing time at 100, 200, 300 msec) experiments were used for structural determination.
Mass spectra were acquired on a microQ-Tof mass spectrometer coupled with an Alliance HPLC (Waters, Milford, MA) equipped with an ESI source.
Microorganism and culture conditions
Geobacillus thermantarcticus (DSM 9572) isolated from Antarctic geothermal soil was grown in a flask (2 L) at 60°C on a medium containing xylan at pH 6.0 as previously reported (Nicolaus et al. 1996). Thermoanaerobacterium thermostercoris strain BUFF was grown at 60°C in a 40 L fermentor (B. Brown Biotech International Micro DCU400) as previously reported (Romano et al. 2010). Thermotoga neapolitana DSM 4359 obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) was grown at 80°C in a 40-L fermentor (B. Brown Biotech International Micro DCU400) as previously reported (Tramice et al. 2007).
Microbial growth was monitored turbidimetrically at 540 nm and cells were harvested in late exponential growth phase and collected by continuous centrifugation using an Alpha-Laval centrifuge and then pelleted by centrifugation at 10,000 rpm for 30 min and frozen.
Preparation of crude enzymatic extracts
Thermoanaeorbacterium thermostercoris (CFE-TT protein 11.4 mg/ml), and Thermotoga neapolitana cell free extracts (CFE-TN protein 34.4 mg/ml) were prepared in acetate buffer 50 mM pH 5.5 disrupting the cells by usual techniques (freeze-thawing, lysozyme 100 μg/ml). Geobacillus thermantarcticus extracellular suspension (ES-GTM1, protein 0.37 mg/ml) was prepared by adding ammonium sulfate to the cell-free broth (1 L) to 80% saturation. The precipitate was recovered by centrifugation (10,000 g, 1 h, 4°C), dissolved in 50 mM sodium acetate buffer (pH 5.6) and dialyzed overnight against the same buffer.
Arundo donax was grown under Mediterranean climatic conditions: during the growth period (April-November 2012) rainfall was 355 mm and Reference Evapotranspiration was 923 mm and thus water deficit was very severe (568 mm). Rhizomes of A. donax were harvested at the experimental farm of the University “Federico II” Naples and were obtained from a 4 year experimental plantation located in the Sele river plain of Campania Region (Southern Italy, 40°61’N, 14°92’E, 30 m a.s.l.). Rhizome samples were harvested in a sample area of 2 m2, and then chipped, dried at 60°C until constant weight and grinded at 4 mm. Successively, the material was grinded in order to obtain a homogenous fine powder (particle size of about 1 mm) by means of a kitchen mixer (Waring).
Preparation and optimization of hemicellulosic substrates from rhizome of Arundo donax
The hemicellulose fraction was solubilized in alkali medium by continuous magnetic stirring at room temperature; different 2% w/v solutions of rhizome of Arundo donax in potassium hydroxide (KOH, 0.5 N, 1 N and 2 N) were used at different time intervals, 24 h, 48 h, 72 h and 96 h. Experiments for the optimization of extraction procedure were conducted in triplicate; briefly, the grinded dry material was suspended in KOH solutions; afterwards the suspension was sieved (0.5 mm screen) and centrifuged at 10,000 rpm for 40 min at 4°C. The supernatant was treated with same volume of cold ethanol 96% (v/v) added drop wise under stirring. The alcoholic suspension was stored at −20°C overnight and then centrifuged at 10,000 rpm for 1 h at 4°C. The pellet was solubilized in hot distilled water, cooled at room temperature, the pH was adjusted to 7.0 with HCl and dialyzed against distilled water using Spectrapore dialysis tubes (12,000-14,000 MW cut-off), and finally freeze-dried. The lyophilized material (RK72h, RK48h, RK24h) was weighed using a Mettler Toledo analytical balance and the yield was expressed in weight (%, w/w) with respect to the initial dry biomass.
Hydrolysis reactions were conducted using various biocatalysts and hemicellulosic material prepared from A. donax (RK72h) as follows: Thermomyces lanuginosus xylanase, 22 units were dissolved in 3 ml phosphate buffer 50 mM, pH 7 containing 30 mg substrate. Reaction was performed at 34°C under stirring for 26 h and stopped by enzyme denaturation at 100°C (2 min). Thermotoga maritima endo-1,4-β-xylanase, 57 units were dissolved in 1 ml in acetate buffer 50 mM, pH 5.6 containing 10 mg of substrate. Reaction was performed under stirring at 70°C, 24 h and stopped by cooling. Cell free extract (CFE-TT) from Thermoanaeorbacterium thermostercoris (proteins: 11.4 mg/ml), was added to the needed amount of substrate (4 mg/ml of reaction) in acetate buffer 50 mM, pH 5.6. Reaction was performed at 50°C for 24 h under stirring. Cell free extract (CFE-TN) from Thermotoga neapolitana (proteins: 34.4 mg/ml), was added to the needed amount of substrate (4 mg/ml of reaction) in acetate buffer 50 mM, pH 5.6. Reaction was performed at 90°C for 24 h under stirring. Extracellular suspension of Geobacillus thermantarcticus 8 ml, ES-GTM1b, (proteins: 0.37 mg/ml) was reacted with 40 mg of substrate solubilized in 2 ml phosphate buffer 50 mM, pH 6, at 70°C for 24 h. In a different condition, ES-GTM1a, 1.5 ml were reacted with 20 mg of substrate solubilized in 4 ml phosphate buffer 50 mM, pH 6, or at 70°C for 24 h.
Transglycosylation reactions were performed using various biocatalysts as follows: 20 mg of A. donax (RK72h) in 1 ml of appropriate buffer/acetonitrile (87:13 v/v) containing 19.6 mg of 9-fluorene methanol; the reactions started by adding the different biocatalysts (57 U Thermotoga maritima endo-1,4-β-xylanase, at 70°C, 24 h; 500 μL Thermotoga neapolitana cell free extract (CFE-TN) at 70°C, 24 h; 500 μL cell free extract (CFE-TT) from Thermoanaeorbacterium thermostercoris at 50°C, 24 h; 4 ml of extracellular suspension Geobacillus thermantarcticus (ES-GTM1) at 70°C, 24 h) under stirring.
Monitoring of reactions, purification and analysis of products
Hydrolyses reactions were monitored by TLC in solvent A: n-BuOH/AcOH/H2O 6:2:2 v/v, or B: EtOAc/AcOH/2-propanol/HCOOH/H2O, 25:10:5:1:15 by vol, whereas transglycosylation reactions were monitored in solvent C: EtOAc/MeOH/H2O 70:20:10 by vol. Chemical determination of reducing sugars and enzymatic determinations of glucose and xylose were conducted in triplicate. Hydrolysis reaction mixture was concentrated under vacuum and loaded onto a Biogel P-100 column (1 × 35 cm) equilibrated with water and eluted at 0.1 ml/min with water. A second Biogel P-2 column (1 × 47 cm) was performed eluting at 0.3 ml/min with water. The fractions containing the carbohydrates were pooled and freeze-dried. Purified material was subjected to NMR spectroscopy. The reactions of transglycosylation were stopped by cooling and immediately subjected to reverse-phase column chromatography (Merck Lobar RP-18) eluting with water, and then with methanol thus efficiently separating total chromophoric xylosylated fraction and unreacted acceptor in methanol fraction from free saccharides in water. The methanolic fractions were collected and analysed by ESI-MS.
Monosaccharides composition of Arundo donax polysaccharides was determined by GC-MS of acetylated methyl glycosides (Silipo et al. 2012). Methanolysis was performed in 1.25 M HCl in methanol. In brief, sample (1.5 mg) was solubilized in anhydrous 1.25 M HCl/MeOH (1 ml) and then heated at 80°C overnight. Subsequently, air-dried samples were acetylated with acetic anhydride (50 μl) and pyridine (50 μl) at 100°C for 30 min. Then, samples were dried and washed with methanol in order to evaporate pyridine and dissolved in acetone (400 μl) and 1 μl was used for GC-MS analysis. All GC-MS runs were conducted on a Hewlett-Packard Series II Plus gas chromatograph (column: 30 m × 0.25 mm HP-5MS Hewlett-Packard Co.) coupled with a Hewlett-Packard 5989B mass spectrometer. Electron voltage was set at 70 eV and He was used as gas carrier at a costant flow rate of 1 ml/min. The temperature program used was 150°C for 3 min; from 150 to 280°C at 10°C/min; 280°C for 20 min.
Antimicrobial activity was carried out using liquid culture of E. coli (DSM 498), B. subtilis subsp. spizizenii (DSM 347), and M. luteus (DSM 348). The MIC was determined by a serial dilution, in duplicate, starting from 100 μg/mL to 0.01 μg/mL. Bacterial and yeast growths were observed after 48 h of incubation. Cytotoxic activity was evaluated by the brine shrimp (Artemia salina) test in triplicate with appropriate amounts of samples dissolved in DMSO (1% final volume) to reach final concentrations of 100, 10 and 1 μg/ml, using 10 freshly hatched larvae suspended in 5 ml of artificial seawater (Meyer 1982). Briefly, for each dose tested, surviving shrimps were left at RT and counted after 24 h, and the data statistically analyzed by the Finney program (Finney 1971), which affords LD50 values with 95% confidence intervals.
Free-radical scavenging activity was evaluated on pre-treated grinded biomass in DMSO and on RK72h dissolved in H2O at a concentration of 20 mg/mL, and assayed for DPPH test (Blois 1958). Different amounts (10, 50, 100 and 250 μL) of these solutions were added to 0.7 mL of DPPH in MeOH (6 mg/50 mL; 0.1 mM final concentration) and adjusted to 2 mL final volume with MeOH. The absorbance at 517 nm was determined after 30 min at room temperature and the percent of free radical inhibition was calculated. Fucoidan (Sigma) was used as standard. Free radical scavenging activity of samples was estimated as % inhibition of free radical DPPH. Antioxidant activity of both samples was also determined by DMPD method (Fogliano et al. 1999). The reaction mixture contained 1 mM DMPD, 0.1 mM ferric chloride in acetate buffer 0.1 M (pH 5.25) in a total volume of 1 mL. The assay temperature was 25°C. The reaction was monitored at 505 nm until absorbance became stable at a value of 0.900 ± 0.100. Samples were dissolved in H2O at a concentration of 20 mg/mL. According to the method, the antioxidant activity of samples was carried out in triplicate on main solution (20 mg/mL) and on its diluted solutions 1:2, 1:5, 1:10. Then, 250 μL of solutions were added to the reaction mixture and the decrease in absorbance, which is proportional to the DMPD+ quenched, was determined after 20 min at room temperature. Fucoidan was used as standard. The antioxidant activity was reported as % inhibition of radical cation DMPD.