Sample collection and metagenomic library construction
Paddy soil was collected from Liaoning province (41°07′03”N 122°03′09”E) of China in October 2010. The total microbial DNA was extracted using SoilMaster™ DNA Extraction Kit (Epicentre, Madison, WI) according to the manufacturer instructions. The metagenomic library was constructed using CopyControl™ Fosmid Library Production Kit (Epicentre, Madison, WI) according to the manufacturer’s instructions. DNA products were examined by agarose electrophoresis, and the fragments within the sizes of 25–35 kb were recovered for an end-repair reaction, and ligated into pEpiFOS-1 fosmid vector prepared in the kit. In vitro packaging was performed with a MaxPlax lambda packaging extract kit (Epicentre, Madison, WI). Finally, the products were infected into E. coli EPI 300 (Epicentre, Madison, WI). The quality of the library was tested by NotI (Promega, Madison, WI) digestion of the prepared fosmids plasmids, and colony counting was carried out by automatic bacteria counter (Shineso Science & Technology Co., LTD, China).
Cellulase screening and gene cloning
The metagenomic library was screened using the substrate carboxymethylcellulose (CMC, Sigma, St. Louis, MO, USA) for CMC hydrolysis genes. The transformed E. coli EPI 300 was inoculated on CMCase screening agar containing 1.0 % tryptone, 0.5 % yeast extract, 1.0 % NaCl, 0.5 % CMC, 100 µg/mL chloramphenicol, and 0.2 mM isopropy-β-D-thiogalactoside (IPTG). The screening agar was incubated at 37 °C for 24 h. After incubation, the plates were stained with 0.2 % Congo-red for 20 min [17]. The CMCase positive clones were screened out by a hydrolysis zone around the bacterial colony. CMCase positive plasmids were enriched with fosmid autoinduction solution and purified by FosmidMAX™ DNA purification Kit (Epicentre, Madison, WI). For subcloning, the plasmid DNA of CMCase positive clone was digested by NotI, then recovered for further digestion by Sau3AI (Promega, Madison, WI), and ligated into a pBlueScript SK(+) vector (Stratagene, La Jolla, CA). The subclone of CMCase positive transformant was screened further, and the insert DNA sequencing was performed on an Applied Biosystems DNA sequencer, model ABI PRISM 377 (Invitrogen, Shanghai).
Sequence analysis and secondary structure prediction
Nucleotide sequence translation, and the possible ORF, signal peptide, theoretical pI, and Mw predictions were performed at online programs (ExPASy and SignalP 3.0) [18, 19]. The conserved region analysis and comparison of sequence identity to the related cellulase genes were performed by BlastX (http://www.ncbi.nlm.nih.gov). Sequences of complete cellulase proteins retrieved from the GenBank database were aligned using the CLUSTAL_X software and the alignments were corrected manually [20]. Phylogenetic analysis was performed by neighbor-joining algorithm using MEGA v5.0, and the topology of the phylogenetic tree was assessed by the bootstrap analysis based on 1000 replications. The circular dichroism (CD) spectroscopy was carried out to estimate the secondary structure of the purified recombinant protein. The CD spectra were determined at room temperature with a JASCO J-810 spectrometer (JASCO Japan), and the protein concentration was 10 µM in Tris–HCl buffer [21]. The secondary structure of the protein was predicted by the online program Protein Homology/analog Y Recognition Engine v2.0 and the online tool K2D2 [21, 22].
Umcel9y-1 gene amplification, overexpression, and purification
Umcel9y-1 gene of the screened subclone was amplified by PCR using the LA PCR™ Kit Ver.2.1 (Takara, Dalian). A primer pair of 5′-GACACCCATGGGCAGCAGCCATCATCATCATCATCAC-3′ (forward primer) and 5′-GTGTCCATATGTCACATTGTTGGAAGCAA-3′ (reverse primer) was employed in the PCR amplification, and the restriction sites of NcoI and NdeI were introduced and underlined. The PCR conditions were 1 min at 94 °C, followed by 30 cycles of 10 s at 95 °C, 35 s at 58 °C, and 5 min at 72 °C. The PCR fragments was first ligated into pUC57 (Sangon, Shanghai), then excised by NcoI and NdeI, ligated into pET-15b(+) vector (Novagen, San Diego, CA), and transformed into the E. coli BL21 CodonPlus™ (DE3) strain. The transformant cells were incubated in Luria-Bertani medium (containing 50.0 µg/mL kanamycin) at 15, 22, and 28 °C for appropriate temperature assay. Until the cell density of OD600 reached 0.6, the broth was induced by adding 0.1 mM IPTG and followed by additional 5 h incubation. His-tagged recombinant protein in cell disruption (precipitant) was purified by affinity chromatography with nickel-nitrilotriacetic acid agarose resin (Ni–NTA, Qiagen, CA). After sample loading, His-tagged recombinant protein was purified by washing buffer (2 M Urea, 50 mM Tris, 2 mM DTT, 10.0–50.0 mM imidazole, pH 8.0), and collected by elusion buffer (2 M Urea, 50 mM Tris, 2 mM DTT, 500 mM imidazole, and pH 8.0). Then, the purified protein was de-His-tagged by TEV protease at 37 °C. The de-His-tag protein was further purified by affinity chromatography to remove the hydrolyzed His-tags, and the Ni2+ ion from Ni-NTA resin was eliminated by dialysis. The purity of recombinant protein was determined by SDS-PAGE using ChemiDoc™ XRS+ system (BioRad, CA), and the protein concentration was estimated by the solution absorbance at 280 nm using a molar extinction coefficient [23].
Substrate specificity and kinetic analysis
Cellulase activity was assessed by measuring the amount of reducing sugars released from CMC (or other substrates) at 575 nm using dinitrosalicylic acid (DNS) reagent [12]. Enzymatic reaction was carried out in 2 mL of mixtures (in phosphate buffer, pH 7.0) containing 1.2 mL cellulase solution (0.12 mg/mL) and 0.8 mL 2.5 % CMC (w/v) at optimum temperature for 30 min. One unit (U) of hydrolysis activity was defined as the amount of enzyme to release 1 µmol of reducing sugar per minute. The substrate specificity of recombinant enzyme was assayed according to the standard methods in phosphate buffer (pH 7.0) at 37 °C [15, 16], and all of the substrates were obtained from Sigma-Aldrich. Other than CMC, the polysaccharides of hydroxyethyl cellulose (HEC), laminarin from Eisenia bicyclis, β-D-glucan from barley, xylan from oat spelt and microcrystalline cellulose (MCC), the cellooligosaccharides of cellobiose (G2), cellotriose (G3), cellotetraose (G4), cellopentaose (G5) and cellohexose (G6), and the aryl-β-glycosides of p-nitrophenol-β-D-cellobioside (pNPCel), p-nitrophenyl-β-D-glucopyranoside (pNPGlc), p-nitrophenyl-β-D-galactopyranoside (pNPGal), p-nitrophenyl-β-D-xylopyranoside (pNPXyl), and p-nitrophenyl-β-D-fucopyranoside (pNPFuc) were selected and determined for substrate specificity. The substrate specificity was assayed under the optimal conditions at a final concentration of 1.0 % substrate (w/v) for 120 min (except 8 h for MCC and 15 min for barley glucan). The specific activities were determined under the optimal condition for 30 min at the substrate concentrations of 40.0 mg/mL for CMC; 20.0 mg/mL for xylan, pNPXyl, pNPGlc, pNPCel, and laminarin; and 15.0 mg/mL for HEC and oligosaccharides. The specific activity of barley glucan was assayed under the optimal condition for 15 min at the substrate concentration of 10.0 mg/mL, and MCC was assayed under the optimal condition for 4 h at the substrate concentration of 20.0 mg/mL. To determine the kinetic parameters of recombinant enzyme, reactions were carried out under the optimal condition for 30 min (except 15 min for barley glucan) at appropriate substrate concentrations (4.0–20.0 mg/mL for CMC, HEC, pNPCel, laminarin and oligosaccharides, and 1.0–10.0 mg/mL for β-D-glucan). The reaction rate versus the substrate concentration was plotted, and the kinetic constants K
m
and K
cat
were calculated by a nonlinear regression of the Michaelis-Menten equation with GraphPad PRISM version 5.0 (GraphPad Software, La Jolla, CA).
Biological characterization of recombinant Umcel9y-1
The pH range was determined with appropriate buffers (Citrate buffer, pH 3.0–6.0; Phosphate buffer, pH 6.0–8.0; Tris-HCl buffer, pH 8.0–9.0; and glycine-NaOH buffer, pH 9.0–10.0), and the temperature range was measured from 20 to 90 °C at the interval of 10 °C using the method for enzymatic activity assay [15]. The enzymatic stability at various temperatures and pH were performed as described previously. The recombinant Umcel9y-1 was incubated at various temperatures for 0–90 min and at various pH for 0–96 h, then the residual activities were determined [24]. Effects of metal ions, EDTA, and chemical agents on the enzymatic activity were tested by appropriate concentrations. The final concentration of the divalent metal ions and EDTA were 1.0 mM, respectively, and the concentration of each chemical agent for the reaction mixtures was 10 % (v/v). The halotolerance was determined by measuring residual activity under optimal condition following pre-incubation of recombinant Umcel9y-1 in 1.0–4.0 M NaCl or KCl for 10 days. The enzymatic activity of recombinant Umcel9y-1 in high-salt concentrations was determined by the enzymatic activity assay with a final concentration of 1–4 M NaCl or KCl in reaction buffers [15]. Influences of monosaccharides on recombinant Umcel9y-1 activity (kinetic parameters) toward barley glucan and cellooligosaccharide were evaluated as previously with 100 mM additive to substrate barley glucan, and 200 or 1000 mM additive to cellopentaose [10].
Substrate hydrolysis products and transglycosylation assay
The hydrolysis products of barley glucan and cellooligosaccharides (G6) by recombinant Umcel9y-1 were detected by high-performance liquid chromatography (HPLC) as described previously [10, 25]. Briefly, hydrolysis was carried out in 1 mL of mixture (in phosphate buffer, pH 7.0) containing 0.6 mL cellulase solution (0.12 mg/mL) and 0.4 mL substrate (50 mg/mL). The hydrolysis was carried out for 24 h on barley glucan and 30 h on G6 at 37 °C. Transglycosylation activity of recombinant Umcel9y-1 was determined in 20 µL mixtures (phosphate buffer, pH 7.0) containing 10 mM pNPGlc, 100 mM glucose, and 0.05 µg recombinant protein. Other than the standard mixture, the reaction system contain 1000 mM glucose was further evaluated. Transglycosylation products were analyzed using a high-performance anion exchange chromatograph with pulsed amperometric detection (HPAE-PAD; Dionex ICS-3000, Sunnyvale CA) as described as Uchiyama et al. [10].
Statistical analysis
Unless specified otherwise, all assays in this study were performed in triplicate. Data are presented as mean ± SD. Statistic analyses were assessed by Student’s t test. The p values <0.05 were considered statistically significant.