chi18H8 cDNA sub-cloning
The nucleotide sequence of the chitinase Chi18H8 gene was previously deposited in the GenBank database under the accession number KC763366 . The cDNA encoding for the chitinase was sub-cloned into the pET24b(+) expression plasmid (kanamycin resistance; Novagen Inc., Madison, USA) by using the primers chiEcoF (5′-ATAAAGAATTCCATGCGCCAGCTCACGCTTCTC-3′) and chiXhoR (5′-ATAAACTCGAGCTAATTGCCCCTATGCAGACTGG-3′), containing the underlined restriction sites for EcoRI and XhoI, respectively. The plasmid pGEX-6P-3::chi18H8, previously prepared , was used as DNA template. E. coli DH5α (Invitrogen-Life Technologies, Carlsbad, USA) was used as host for the sub-cloning procedures. The construct, following its control by DNA sequencing (BMR Genomics, Padua, Italy), was transformed into E. coli BL21 Star™(DE3) (Invitrogen-Life Technologies). Recombinant E. coli strains were maintained on Luria-Bertani broth (LB, Miller’s modification; Sigma-Aldrich, St. Louis, USA) agar plates supplemented with 50 µg/mL kanamycin.
All medium components and reagents were from Sigma-Aldrich, unless otherwise stated. Protein expression was carried out in the following media, supplemented with 50 µg/mL kanamycin: LB; terrific broth (TB); super broth (SB: 32 g/L tryptone, 20 g/L yeast extract, 5 g/L NaCl); autoinduction media A and B. Autoinduction medium A composition was based on . Autoinduction medium B included: 10 g/L tryptone, 5 g/L yeast extract, 3.3 g/L (NH4)2SO4, 6.8 g/L KH2PO4, 7.1 g/L Na2HPO4, 0.5 g/L glucose, 2 g/L α-lactose, 0.15 g/L MgSO4, 2 mg/L CaCl2, 2 mg/L MnSO4 × H2O, 2 mg/L ZnSO4, 2 mg/L CoCl2, 2 mg/L CuCl2 × 2H2O, 2 mg/L NiCl2, 2 mg/L NH4MoO4, 2 mg/L FeCl3. Trace element (MgSO4, CaCl2, MnSO4 × H2O, ZnSO4, CoCl2, CuCl2 × 2H2O, NiCl2, NH4MoO4, FeCl3) stock solutions were sterilized by filtration (0.2 µm) and stored at 4 °C.
Starter cultures were prepared from a single recombinant E. coli colony inoculated in 10 mL LB medium supplemented with 50 µg/mL kanamycin, grown overnight at 37 °C and 200 revolutions per minute (rpm). Baffled 300 mL Erlenmeyer flasks containing 50 mL of the different media were inoculated with the starter culture (initial OD600nm = 0.1) and further incubated as above. For LB, TB and SB media, protein expression was induced by adding 0.4 mM isopropyl β-d-thiogalactopyranoside (IPTG) to cells at early- or late-exponential growth phase. After induction, cells were cultured at various temperatures (37, 25 or 20 °C, respectively) at 200 rpm and harvested by centrifugation (1900×g for 30 min at 4 °C) at different time intervals. Following centrifugation, total proteins in the supernatants (i.e. the cell-free fermentation broths) were concentrated by 10% (v/v) trichloroacetic acid precipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, see below). In parallel, cell pellets were sonicated on ice (3–5 cycles of 30 s each, with a 30-s interval, using a Branson Sonifier 250, Danbury, USA) in phosphate buffer saline (PBS) pH 7.3 (140 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4) containing 10 µg/mL deoxyribonuclease (DNase), 0.19 mg/mL phenylmethylsulfonylfluoride (PMSF) and 0.7 mg/mL pepstatin. Soluble (cytoplasmic) and insoluble (containing membranes and IBs) cell fractions were then separated by centrifugation at 20,000×g for 1 h at 4 °C. Insoluble fractions were re-suspended in a volume of PBS equal to the corresponding cytoplasmic soluble fraction (2 mL/g cell) for successive SDS-PAGE analyses. In every fraction, protein concentration was determined by the Biuret assay .
Flask cultures of recombinant E. coli cells, grown overnight in LB medium supplemented with 50 µg/mL kanamycin, were used to inoculate (initial OD600nm = 0.1) 2 L Erlenmeyer flasks (containing 750 mL medium), or 3 L P-100 Applikon glass reactors (Applikon Biotechnology, Delft, The Netherlands) equipped with a AD1030 biocontroller and AD1032 motor (containing 2 L medium), or 30 L Bioengineering (Bioengineering AG, Wald, Switzerland) stirred fermenter (containing 27 L medium). The medium used throughout was LB medium supplemented with 50 µg/mL kanamycin. 2 L Erlenmeyer flasks were incubated at 37 °C and 200 rpm. Growth in fermentors was conducted at 37 °C and stirring at 500 rpm for 3 L bioreactor or at 300 rpm for the 30 L bioreactor, respectively, at constant 1.0 vvm aeration rate and pressure control set at 0.5 bar. Dissolved oxygen (measured as % of the initial pO2 value) and pH were monitored using an Ingold polarographic oxygen electrode and a pH meter, respectively. Foam production was monitored through an antifoam sensor and controlled by adding Antifoam SE-15. When cell culture reached an OD600nm = 0.6, protein production was induced by adding 0.4 mM IPTG: cultivation was then prolonged at 20 °C for further 24 h.
Recovery of IBs and solubilization of Chi18H8
Escherichia coli cells were harvested by centrifugation at 3220×g for 20 min, washed with sodium chloride-Tris-EDTA (STE buffer: 10 mM Tris–HCl pH 8.0, 1 mM EDTA [ethylenediaminetetraacetic acid], 100 mM NaCl). The different protocols for IB recovery and solubilization are described in Additional file 1: Table S1. In the case of the acid solubilization method, after centrifugation, the cell pellet was re-suspended in 50 mM Tris–HCl pH 8.0, 25% (w/v) sucrose, 1 mM EDTA, and incubated for 30 min at room temperature under vigorous shaking. After sonication on ice (6 cycles of 30 s each, with a 30-s interval), 0.2 M NaCl, 1% (w/v) sodium deoxycholate (DOC) and 1% (v/v) Nonidet P-40 were added. The sample was further incubated as above and centrifuged (20,000×g at 4 °C for 30 min). The pellet was washed with 1% (v/v) Triton X-100 and 1 mM EDTA, followed by centrifugation at 12,000×g at 4 °C for 10 min; the procedure was repeated twice. IBs were then washed twice with 10 mL/g cell deionized water, with centrifugation at 12,000×g at 4 °C for 10 min in-between, and stored overnight at −20 °C. To solubilize Chi18H8 from IBs, the frozen pellet was re-suspended in 10 mL/g cell of 10 or 100 mM HCl or lactic acid and incubated at 37 °C and 200 rpm for 5 h. Insoluble material was removed by centrifugation at 1900×g at 4 °C for 5 min. The solubilized protein was then dialyzed overnight against 100 mM sodium acetate buffer pH 5.0 or 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 5.6.
Enzyme samples following dialysis were used for testing different chromatographic methods, described in details in Additional file 1: Table S2. Chi18H8 purification/enrichment was achieved by loading the protein sample after dialysis against 100 mM HEPES pH 5.6 onto the weak anionic exchanger Diaion WA11 resin (Resindion s.r.l., Milan, Italy), previously activated with methanol/water (1:1) and then equilibrated with 100 mM HEPES pH 5.6. Pilot experiments were performed with 1 mL resin (wet volume) or, for large scale purification, with 40 mL of WA11 resin (in a 4.5 cm diameter Amicon column loaded at a flow rate of 20 mL/min). The chromatography, run under hydrophobic interaction (HIC) conditions, allowed to fully recover the Chi18H8 protein in the flow-through, whereas other proteins retained by the WA11 resin were then recovered by an isocratic elution using 50% (v/v) ethanol in 100 mM HEPES pH 5.6. The Chi18H8 concentration was estimated using the theoretical extinction coefficient at 280 nm (77,015 M-1cm-1), based on the amino acid sequence of the protein.
SDS-PAGE electrophoresis and zymogram analysis
Chi18H8 size, production and solubilization were analyzed by SDS-PAGE with 12% (w/v) polyacrylamide gels  and estimated by densitometric analysis (Quantity One, Bio-Rad Laboratories, Hercules, USA) using known amount of His6-glycine oxidase (His6-GO) from Bacillus subtilis  as a standard. Chitinolytic activity was detected through zymogram analysis (semi native-PAGE) using 10% (w/v) polyacrylamide gels containing 0.7 mg/mL of carboxymethyl-chitin-remazol brilliant violet (CM-chitin-RBV) (Loewe Biochemica, Suerlach, Germany), as described in . Briefly, the protein samples were diluted in a sample buffer lacking any reducing agent and incubated for 10 min at room temperature. Electrophoresis was conducted at 4 °C in Tris-glycine-SDS running buffer according to standard running conditions. The gel was rinsed twice in 2.5% (v/v) Triton X-100 for 30 min at room temperature to remove SDS and incubated in 100 mM sodium acetate pH 5.0 at 37 °C until the appearance of clear zones indicating chitinolytic activity. The chitinase from Trichoderma viride (Sigma-Aldrich) was used as positive control.
Chi18H8 activity assay
Chi18H8 activity was assayed with the fluorimetric chitooligosaccharide analogues 4-methylumbelliferyl N-acetyl-β-d-glucosaminide (4-MU-GlcNAc), 4-methylumbelliferyl N,N’-diacetyl-β-d-chitobioside (4-MU-(GlcNAc)2) and 4-methylumbelliferyl N,N’,N’’-triacetyl-β-d-chitotrioside (4-MU-(GlcNAc)3) as described previously . Unless otherwise stated, Chi18H8 activity was assayed at 37 °C in 100 mM sodium acetate pH 5.0 . Chitinolytic activity was also determined on colloidal chitin, prepared from chitin flakes from shrimp shells (Sigma-Aldrich), by the colorimetric method previously described . One unit (U) of Chi18H8 activity was defined as the amount of enzyme that released 1 μmol of 4-MU or GlcNAc per min at 37 °C.
Effect of pH and temperature on Chi18H8 activity
pH influence on Chi18H8 activity on the substrate 4-MU-(GlcNAc)2 was determined by the fluorimetric assay in the following buffers (each 100 mM): glycine–HCl (pH 3.0), sodium acetate (pH 4.0 and 5.0), sodium phosphate (pH 6.0 and 7.0), Tris-HCl (pH 8.0) and sodium pyrophosphate (NaPPi, pH 9.0). Optimal temperature for the enzyme activity was determined by the same fluorescent assay, performing the reaction at various temperatures (from 5 to 70 °C). Stability of the enzyme was tested by incubating the recombinant protein at 30 °C at different pHs (in 100 mM sodium acetate pH 5.0 or 100 mM sodium phosphate pH 6.0 and 7.0): after different incubation times (from 0 to 144 h), the residual chitinolytic activity was assayed on 4-MU-(GlcNAc)2.
Effect of metal ions, organic solvents, detergents and other compounds on Chi18H8 activity
The effect of metal ions [Ca2+ (CaCl2 × 2H2O), Cu2+ (CuCl2 × 2H2O), Fe3+ (FeCl3 × 6H2O), K+ (KCl), Mg2+ (MgCl2 × 6H2O), Mn2+ (MnCl2 × 4H2O), Ni2+ (NiCl2 × 6H2O), NH4 (NH4Cl), Zn2+ (ZnCl2), Co2+ (CoCl2 × 6H2O)], reducing agents [dithiothreitol (DTT), 2-mercaptoethanol], the chelating agent EDTA, detergents [SDS, Triton X-100, Tween-20, DOC, N-lauroylsarcosine (NLS), Nonidet-40], organic solvents [ethanol, methanol, propanol, dimethyl sulfoxide (DMSO)], sugars (GlcNAc, chitobiose) and salt (NaCl) on Chi18H8 activity was investigated by adding each compound to the fluorimetric assay mixture. Final concentrations were: 20 mM for metal ions, EDTA and 2-mercaptoethanol, 10 mM for sugars and DTT, 10% (v/v) for organic solvents, and 1% (w/v) for detergents. In each case the residual activity was compared to the activity without additional compounds, set as 100%.
Far-UV CD spectra were recorded at 15 or 30 °C using a Jasco J-715 spectropolarimeter (Jasco, Cremello, Italy) equipped with temperature control, in the 190–250 nm wavelength range. Measurements were carried out in quartz cuvettes of path length 1 mm, employing a protein solution at 0.2 mg/mL in 10 mM HEPES pH 5.4, and corrected for buffer contribution. Secondary structure composition was calculated from deconvolution of the CD spectra with the program k2d3 (http://k2d3.ogic.ca//index.html) . Binding of Ca2+ to purified Chi18H8 was investigated by recording the far-UV CD spectrum during the titration of the apoprotein form of the enzyme (obtained by incubation for 30 min with 20 mM EDTA), with increasing concentrations of CaCl2 (5, 10, 100, 1000 and 10,000 μM). The change in CD signals at 219 nm versus CaCl2 concentration were fitted to a classical 1:1 complex saturation equation to calculate the dissociation constant, Kd.
Determination of mode of action
A chitin-like substrate, i.e. a water-soluble high molecular weight chitosan with a degree of acetylation of 63%, was used as substrate for Chi18H8 to determine the enzyme’s mode of action as previously described . The chitosan (30 mg) was dissolved in 3 mL of 80 mM sodium acetate, pH 5.5. To start the depolymerization reaction, 2.5, 7.5 or 26 µL of Chi18H8 (1.16 mg/mL) were added to 1 mL substrate. After incubating the mixture at 37 °C for 30 min, the reaction was stopped by adjusting the pH to 2.0 and boiling for 10 min. The reaction mixture containing the oligomers was injected and separated on three XK 26 columns in series, packed with Superdex™ 30 (Amersham Pharmacia Biotech, Piscataway, USA) using a refractive index (RI) detector (Shimadzu Schweiz GmbH, Reinach, Switzerland), as previously described .
Antifungal activity assay
Chi18H8 antifungal activity was evaluated by growth repression assays, in triplicate, on Fusarium graminearum ATCC 46779, Rhizoctonia solani ATCC 10183 and Botrytis cinerea ATCC 26943. Plate assays were carried out by centrally inoculating an agar plug, harboring an actively growing fungal culture, in 9-cm diameter Petri plates containing diluted potato dextrose agar (PDA). Sterilized paper discs (Munktell Filter ab, Falun, Sweden) 5 mm in diameter were soaked in 1000, 500, 100 or 10 µg/mL, respectively, of the solubilized Chi18H8 (in 100 mM sodium acetate, pH 5.0) and placed 25 mm around the fungal plug. A liquid assay system was also used. Wells (2 mL cellplate, Biofluidfocus, Largo, USA) containing fungal PDA agar plugs and 200 μL potato dextrose broth (PDB) were added with Chi18H8 (in 100 mM sodium acetate, pH 5.0) to a concentration of 1000, 100 or 10 µg/mL. Boiled enzyme in the same buffer was used as negative control in both assays. The systems were incubated at 23 °C in darkness for 48 h, and longer for F. graminearum due to slower mycelial growth rate. Growth of fungal hyphae was monitored daily using a microscope. For the liquid assay, dry weight biomass of the fungal plugs was measured seven days after inoculation.