Chemicals
The p-nitrophenyl-β-d-xylopyranoside (pNPX), p-nitrophenyl-β-d-glucopyranoside (pNPG), beech wood xylan, N-ethylmaleimide (NEM), iodoacetate, phenyl methylsulfonyl fluoride (PMSF), diethyl-pyrocarbonate (DEPC), 1 ethyl-3-(3 dimethyl aminopropyl) carbidiimide (EDAC), 2-4-6 trinitrobenzenesulfonic acid (TNBS), 5-bromosuccinimide (NBS), N-acetylimidazole (NAI), citraconic anhydride, acetic anhydride, phenyl glyoxal, HEPES and MES, QAE-Sephadex A-50, Sephacryl-200 Coomassie Brillient Blue G-250, Bromophenol Blue were obtained from Sigma-Aldrich, St. Louis, USA. SDS-PAGE markers were purchased obtained from Invitrogen. All other chemicals were commercially sourced and used without further purification.
Microbial strain, growth media and enzyme production
Pseudozyma hubeinsis NCIM 3574 was obtained from NCIM Resource Center, CSIR-National Chemical Laboratory, Pune, India. It is also deposited in NCYC, UK with an accession number NCYC 3431. The strain was maintained on MGYP agar medium consisting of 0.3 % malt extract, 1 % glucose, 0.3 % yeast extract, 0.5 % peptone and 2 % agar and it was sub-cultured once in every 15 days. The fermentation medium used for enzyme production consisted of 0.05 % NaNO3, 0.05 % KCl, 0.05 % MgSO4, 0.02 % K2HPO4, 0.1 % yeast extract, 0.5 % bacto-peptone and 2 % xylan. The initial pH of the medium was adjusted to 5.5 prior to sterilization. For enzyme production, the submerged fermentation (SmF) was carried out in 250-mL Erlenmeyer flasks with 70 mL of the fermentation medium. The flasks were inoculated with 5 % inoculum prepared in MGYP liquid medium and incubated at 27 °C with shaking at 170 rpm. The cell growth was harvested after 120 h by centrifugation (7000×g, 15 min) and the supernatant was used as a source of crude enzyme. To see the effect of temperature on enzyme production, the SmF was carried out at different temperatures (25–30 °C) and samples were removed at different time intervals, centrifuged and analyzed for enzyme activity.
Analytical methods
β-Xylosidase (β-d-xylan xylohydrolase, EC 3.2.1.37) activity was estimated using pNPX as substrate in 50 mM citrate buffer, pH 4.5. The total 1 mL of reaction mixture consisted of 0.9 mL of pNPX (0.5 mg mL−1) and 0.1 mL of suitably diluted enzyme. The reaction was initiated by the addition of enzyme followed by incubation at 60 °C for 30 min. The reaction was terminated by the addition of 2 mL of 2 % sodium carbonate and the liberated p-nitro phenol was measured at 410 nm. One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 µmol of p-nitro phenol from the substrate. Protein was determined according to Lowry method (Lowry et al. 1951) with bovine serum albumin as standard. Glycoprotein content of the purified enzyme was determined by the phenol–sulfuric acid method (Dubois et al. 1956) with d-mannose as the standard.
Native polyacrylamide gel electrophoresis and zymogram of β-xylosidase
For zymogram staining, the crude enzyme preparation was fractionated by native polyacrylamide gel electrophoresis (PAGE) using 10 % acrylamide as resolving gel and 4 % stacking gel (Laemmli 1970). The β-xylosidase activity in the gel was detected by developing zymogram against 10 mM 4-methylumbelliferyl-β-d-xyloside as substrate prepared in 50 mM sodium citrate buffer (pH 4.5). Upon completion of electrophoresis, the gel was immersed in substrate solution for 45 min at 50 °C in the dark. The β-xylosidase bands in the gel were visualized under UV light using Gel Documentation system (Syngene).
Purification of β-xylosidase
The fermented broth was centrifuged at 7000×g for 10 min and the supernatant was concentrated by ammonium sulfate precipitation at 90 % saturation at 4 °C with constant stirring and left overnight. The concentrated crude extract (5 mL) was loaded onto a QAE-Sephadex A-50 column (30 × 2.5 cm) pre-equilibrated with 20 mM glycine–NaOH buffer (pH 8.0). The column was then washed with the same buffer to confirm that the flow-through fractions showed no activity. The bound proteins were then eluted with 0.3 M NaCl at a flow rate of 1 mL min−1 and the fractions (3.0 mL) with β-xylosidase activity were pooled and then concentrated. The concentrated fraction was dialyzed extensively against the 10 mM glycine–NaOH buffer (pH 8.0). The dialyzed fraction was freeze dried and dissolved in minimal volume of 10 mM glycine–NaOH buffer (pH 9.0). This concentrated fraction was applied to Sephacryl S-200 column (1.5–110 cm) previously equilibrated with 10 mM glycine–NaOH buffer (pH 8.0) and the fractions were collected at a flow rate of 0.2 mL min−1. Fractions (1.8 mL) showing β-xylosidase activity were pooled together, concentrated by freeze drying and the purified concentrated enzyme was stored at 20 °C till further use.
Enzyme characterization
The molecular mass of PhXyl was determined by 10 % SDS–PAGE. The molecular mass of the native enzyme was determined by matrix assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrophotometry, using Voyager DE-STR (Applied Biosystems, USA) equipped with a 337 nm nitrogen laser. The matrix was prepared in deionized water containing sinapinic acid (10 mg mL−1), 50 % acetonitrile and 0.1 % TFA. The β-Xylosidase was mixed with matrix (1:1) and 2 µL of the sample was spotted on plate, dried at room temperature.
The optimum pH of the enzyme was determined by estimating enzyme activities at 65 °C in 50 mM citrate phosphate buffer at different pH values (2.5–6.0). The pH stability studies were performed by incubating the enzyme in 50 mM buffer systems with different pH values ranging from 2.0 to 9.0 (KCl–HCl buffer, pH 2.0; citrate phosphate buffer, pH 2.5–6.0; phosphate buffer, pH 7.0; glycine NaOH buffer, pH 8.0–11.0) at 30 °C. The residual enzyme activity was then assayed under standard assay conditions. The optimal temperature of the enzyme was determined by performing the enzyme assays at various temperatures (40–80 °C) in 50 mM citrate buffer (pH 4.5). Temperature stability of the enzyme was determined by pre-incubating the enzyme in 50 mM citrate buffer (pH 4.5) for 4 h at different temperatures (50–70 °C) followed by measuring the residual activity under standard assay conditions. The effect of heavy metals and EDTA on enzyme activity was determined by performing enzyme assays in presence of respective metal salts and EDTA at varying concentrations (0.1, 1.0 and 10 mM).
Substrate specificity studies were carried out using pNP-β-glucopyranoside and pNP-α-l-arabinopyranoside, pNP-β-xylopyranoside as substrates. The K
m and V
max values of purified PhXyl were determined under standard assay conditions using 0.23–5.52 mM of pNPX as substrate. The constant values were calculated by fitting data to nonlinear regression using Michaelis–Menten equation.
To determine the effect of xylose on catalytic activity, assays were carried out in presence of various xylose concentrations (25–200 mM) using pNPX under standard assay conditions. To confirm the type of inhibition, kinetic constants (K
m and V
max) were determined using different inhibitor concentrations (10, 15 and 20 mM) of xylose at varying pNPX concentrations (0.23–5.52 mM) under standard assay conditions. The effect of ethanol on enzyme activity was studied by incubating the enzyme in presence of ethanol at various concentrations (5–30 %, v/v) and the activity was determined at 40 and 60 °C under standard assay condition. The activity assayed in absence of ethanol was recorded as 100 %.
Chemical modification studies using group specific reagents
Purified PhXyl (5 µg each) was incubated with various amino acid functional group specific reagents in 1 mL of the total reaction mixture. Chemical modification studies were performed under the conditions given in Table 5. After 30 min incubation at 30 °C, residual activity of enzyme samples was determined under standard assay conditions.
Modification of carboxyl residue was performed by incubating β-xylosidase (10 µg) with varying concentrations of EDAC (50–200 mM) in 1 mL of 50 mM MES/HEPES buffer (75:25), pH 6.0 at 30 °C. The control was kept without addition of EDAC. Samples were withdrawn after suitable time intervals and the reaction was terminated by addition of 1 mL of 50 mM citrate buffer, pH 4.5. The residual activity of modified enzyme was determined under standard assay conditions. Tryptophan residues were modified by incubating purified enzyme with increasing concentrations of NBS (0.1–1.0 mM) in 50 mM of sodium citrate buffer, pH 4.5 at room temperature. After 10 min, the aliquots were removed for analysis of residual enzyme activity. Tyrosine residue were modified by incubating purified enzyme with increasing concentration of N-acetyl-imidazole (10–50 mM) in 50 mM Sodium borate buffer, pH 7.6. Substrate protection studies were carried out by incubating the β-xylosidase with excess amount of substrate pNPX for 10 min followed by treatment with corresponding modified reagent. The residual enzyme activity was assayed under standard assay conditions.
Mass spectrometric analysis of the purified protein
Proteins in gel bands were reduced, carboxyamidomethylated and digested with Trypsin Gold (Promega) on a robotic platform for protein digestion (MassPREP station, Waters). Resulting peptides were analysed by ESI–MS/MS after on-line separation on a C18 reversed phase, 75 μm inner diameter, 15 cm column (Jupiter 4 µm Proteo 90 Å, Penomenex, column made in-house, courtesy of David Tooth, UoN). Peptides were delivered via a CapLC HPLC attached to a Q-TOF2 mass spectrometer equipped with a nano-electrospray source (Waters) and operated with MassLynx Version 4.0 acquisition software. ProteinLynxGlobalSERVER software Version 2.1 (Waters) was used to generate a peak list file of un-interpreted fragment mass data which was used to search against all entries in the NCBInr (version 20151016) and SWISSPROT databases using the MASCOT search engine (http://www.matrixscience.com). Carbamidomethylation of cysteine and oxidation of methionine were set as variable modifications. One missed cleavage by trypsin was accepted. Only protein identifications with probability-based MOWSE scores above a threshold of p < 0.05 were accepted.