Plasmids, strains and culture conditions
Escherichia coli DH5α was used in all cloning experiments. The plasmid pPICZαA and wild-type Pichia pastoris X-33 were used for the eukaryotic expression of ArCel5 and mutants, whereas the plasmid pET28a and Escherichia coli BL21 (DE3) were ready for the prokaryotic expression of ArCel5. All the above plasmids and strains were kind gifts from Prof. Zhao (Dalian Institute of Chemical Physics, CAS). Cellulose-gelatinizing strain Arthrobotrys sp. CX1 was previously isolated by the author’s laboratory (Lan et al. 2016) and used in this study for cloning of ArCel5-encoding genes.
Arthrobotrys sp. CX1 was cultivated at 30 ℃ for 8–10 days on the filter paper agar plate (per liter was composed of 2 g yeast extract and 20 g agar in the mineral salt solution to produce mineral salt agar plate, and a piece of Whatman filter paper on the mineral salt agar plate). The mineral salt solution (per liter was composed of 2 g MgSO4·7H2O, 2 g Na2HPO4, 0.2 g FeSO4·7H2O, 0.7 g CaCl2, at pH 6.8–7.0) supplemented with 100 mg of ampicillin and 30 mg of chloramphenicol. Until the translucent paper was observed, a small piece of translucent filter paper (0.5 cm × 0.5 cm) from the colony edge was transferred into 100 mL microcrystalline cellulose medium (per liter was composed of 2 g yeast extract and 10 g Avicel PH101 microcrystalline cellulose in the mineral salt solution). Then Arthrobotrys sp. CX1 was cultivated at 30℃ for 7 days in 250-mL flasks on a rotary shaker incubated at 200 rpm. E. coli strains were grown at 37 ℃ in LB medium (per liter was composed of 10 g tryptone, 5.0 g yeast extract, 10 g sodium chloride at pH 7.0) supplemented with 25 μg/mL zeocin or 100 μg/mL kanamycin if necessary. Pichia pastoris strains were cultured at 30 ℃ in YPD medium (per liter was composed of 10 g yeast extract, 20 g peptone, 20 g dextrose, at pH 6.0), BMGY medium (per liter was composed of 3.4 g YNB, 10 g ammonium sulfate, 10 g glycerol, 10 g yeast extract, 10 g peptone, 100 mL of 1 M phosphate buffer, pH 6.0, and 2 mL of 200 g/L D-biotin), and BMMY medium (identical to BMGY except that it contains 10 mL/L of methanol instead of glycerol).
Analysis of the composition of potential accessory protein by MS-based
Arthrobotrys sp. CX1 was cultured on a filter paper agar plate for 10 days at 30 ℃, and the filter paper samples in the degraded area (transparent) and the undegraded area (opaque) were collected and analyzed by SDS-PAGE. The Micro Protein PAGE Recovery Kit (Sangon Biotech, China) was used to recover differentially expressed proteins. The differentially expressed protein was dissolved in buffer I (8 M urea + 50 mM Tris–HCl, pH 8.0). After that, the proteins were reduced by 20 mM DTT at 60 ℃ for 1 h, after cool down to room temperature, alkylated by 20 mM IAA at room temperature for 40 min in the dark, then diluted to buffer II (1 M urea + 50 mM Tris–HCl, pH 8.0). Trypsin (protease concentration 1 mg/mL) was added with an enzyme to protein mass ratio of 1:20, and the peptides were obtained by incubation at 37 ℃ for 20 h. The tryptic peptides were desalted by the Oasis HLB column (Waters, America) and dissolved in 0.1% FA after vacuum concentration. Peptides were loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific), then peptides were separated by an Acclaim PepMapTM RSLC reversed-phase analytical column. The analysis of peptides by using Q-Exactive mass spectrometer equipped with a nanospray ion source and an Ultimate 3000 RSLC nano System. Using MS/MS data obtained by Proteome Discoverer 2.2.0.388. MS identification was performed as described previously (Sun et al. 2019).
Protein sequence analysis and molecular modeling
Sequence alignment was analyzed by Clustal Omega (Sievers et al. 2011), and the sequence alignment results were output by Espript 3 server (Patrice et al. 2003). The structure of ArCel5 was predicted by using the I-TASSER protein structure homology-modeling online server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). Models were visualized with PyMOL (http://pymol.sourceforge.net/). The molecular weight of the protein is estimated on the tool page of ExPASy (http://www.expasy.org/tools/). The signal peptide of the gene encoding wild-type ArCel5 was predicted by SignalP 4.0 (Petersen et al. 2011). NetOGlyc (http://www.cbs.dtu.dk/services/NetOGlyc/) and NetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/) servers were used to predict glycosylation sites.
Expression and purification of ArCel5 and its mutants
After 7 days of cultivation in microcrystalline cellulose medium, the cells of Arthrobotrys sp. CX1 were collected and washed repeatedly with double distilled water. Total RNA of Arthrobotrys sp. CX1 was isolated using RNAiso Plus (TaKaRa Bio Inc., Dalian, China) according to the manufacturer’s protocol. The RNA was reverse-transcribed into cDNA using a PrimeScript™ One Step RT-PCR Kit Ver.2 (TaKaRa Bio Inc., Dalian, China) according to the manufacturer’s instructions, and the recombinant plasmid was amplified by PCR using PrimeSTAR HS DNA Polymerase (TaKaRa Bio Inc., Dalian, China) and the following primers (Additional file 1: Table S1). The native signal peptide of the ArCel5 was removed from all recombinants. These PCR products of different lengths gene encoding ArCel5 were digested with EcoRI and XbaI and ligated into EcoRI-XbaI-cut pPICZαA to construct recombinant plasmids pPICZαA-ArCel5, pPICZαA-ArCel5-NG, pPICZαA-ArCel5-LD, pPICZαA-ArCel5-D. The PCR product of arcel5 was digested with EcoRI and HindIII and ligated into EcoRI-HindIII-cut pET28a to construct recombinant plasmid pET28a-ArCel5. pPICZαA-ArCel5, pPICZαA-ArCel5-NG, pPICZαA-ArCel5-LD, pPICZαA-ArCel5-D after digested by SacI, linearized recombinant plasmids were transformed into P. pastoris X-33 to express ArCel5 mutants, respectively. As shown in Fig. 5a, pPICZαA-ArCel5 was used to express ArCel5 (Pp-ArCel5), pPICZαA-ArCel5-NG was used to express the nonglycosylated mutant (Pp-ArCel5-NG) that has a linker with mutation all of the Thr residues to Ala, pPICZαA-ArCel5-LD was used to express a mutant with the removal of CBM1 (Pp-ArCel5-LD), pPICZαA-ArCel5-D was used to express a mutant with the removal of CBM1 and linker (Pp-ArCel5-D), the above four plasmids after digested by SacI, linearized recombinant plasmids were transformed into P. pastoris X-33 to express ArCel5 mutants, respectively. pET28a-ArCel5 was transformed into E. coli BL21 (DE3) used to express ArCel5 (Ec-ArCel5). Transformation methods referred to the Invitrogen Pichia pastoris expression manual and pET system operation manual.
For the expression of ArCel5 and its mutants in P. pastoris X-33, the recombinant cells were first grown in 5 mL YPD medium containing zeocin (100 mg/mL), then incubated at 30 ℃ and 200 rpm. After 12 h, 0.5 mL cultures were inoculated to 50 mL BMGY medium in 500 mL shake flasks at 30 ℃ and 200 rpm for 16–18 h until OD600 reached 4.0. Cells were collected by centrifugation at room temperature, resuspended in 200 mL of BMMY medium in a 2-L baffled flask, and then incubated at 30 ℃ for 96 h. Methanol was added every 24 h with the final concentration of 1% (v/v) to maintain induction of ArCel5. The culture supernatants were collected by centrifugation at 8000 g for 30 min at 4℃. The fermentation supernatant was filtered through a 0.22 μm polyethersulfone membrane. The concentrated enzyme solutions were purified by a 10-kDa molecular weight cutoff membrane (Millipore Corporation, USA).
For the expression of ArCel5 in E. coli BL21 (DE3), transformants were grown at 37 ℃ in 200 mL LB medium supplemented with 50 mg/L kanamycin until OD600 reached 0.5 and induced by adding IPTG at a final concentration of 0.5 mM, after which the cells were further cultured at 16 ℃ for 14 h. Cells were harvested by centrifugation, and the cell pellet was suspended and sonicated 15 min on ice for 1 s with 3 s intervals in between with an ultrasonic processor. Following the removal of cell debris by centrifugation, the supernatant was ready for subsequent purification. Subsequently, His-tagged proteins were purified by the Ni–NTA purification system using an AKTA Prime Plus (GE Healthcare) as described previously (Crowe 1994).
Comparative analysis by SDS-PAGE
According to the method described by Laemmli (Laemmli 1970), protein samples were mixed with 5 × loading buffer and incubated at 95 ℃ for 10 min prior to being analyzed on 12% (w/v) polyacrylamide gel. The gel was stained with Coomassie brilliant blue R-250, and the stained protein gel was decolorized with decolorizing solution (5% ethanol, 10% acetic acid). Glycosylation staining used the Pierce™ Glycoprotein Staining Kit (Thermo Fisher Scientific), following the manufacturer’s instructions. Western blot was analyzed as previously reported (Effenberger et al. 2017).
Characterization of binding affinity
The adsorption experiment was carried out in a 2-mL tube. Incubate the accessory protein with 10 mg/mL Avicel and filter paper substrates, which were dissolved in 50 mM acetic acid–sodium acetate buffer (pH 5.0). The protein concentration was 0.1–1 mg/mL, the total volume was 0.5 mL, the adsorption temperature was 25 °C, and the substrate was shaken at 120 rpm for 1 h to ensure the adequate combination of protein and substrate. Centrifuge (5 min, 12,000g, room temperature) and measure the content of unbound protein with Bio-Rad Protein Assay Kit (Bio-Rad, USA). The Langmuir adsorption isotherms of filter paper and Avicel were selected by appropriate fit (R2 ≥ 0.98). To study the role of different domains in binding to cellulose substrate, Pp-ArCel5, Pp-ArCel5-NG, Pp-ArCel5-LD, Pp-ArCel5-D, and cellulose samples were incubated under the same conditions. The adsorbed accessory protein concentration was calculated as the difference between the total protein and the unbound accessory protein concentration. The adsorption isotherm parameters were determined by Langmuir isotherm (Arola and Linder 2016; Eibinger et al. 2016), where B represents the adsorption amount of protein per gram of cellulose (μmol/g), Bmax represents the maximum adsorption amount of protein per gram of cellulose in an equilibrium state (μmol/g), and Kd represents dissociation constant (μmol/L).
Scanning electron microscopy analysis
Scanning electron microscopy (SEM) (JEOL, Japan) was applied to the microstructural changes and surface characteristics of Pp-ArCel5 treatment on filter paper. The filter paper used as control was treated with BSA in 50 mM acetic acid–sodium acetate buffer (pH 5.0). Before SEM evaluation, the dried samples were coated with a thin layer of gold to prevent the sample from becoming charged under the electron beam.
Enzyme assay
The specific activity of the enzymes was measured on carboxymethyl cellulose (CMC), Whatman filter paper, cotton, Avicel PH-101, respectively. All reactions were done in 50 mM acetic acid–sodium acetate buffer (pH 5.0) at 55 ℃ for 30 min, and the total reaction volume was 200 μL. The final concentration of Pp-ArCel5, Pp-ArCel5-NG, Pp-ArCel5-LD, Pp-ArCel5-D used in the assay was 0.1 mg/mL, and substrate concentration was 10 mg/mL. The release of reducing sugar was measured by the 3,5-dinitrosalicylic acid (DNS) method with glucose as a standard (Miller 1959). One unit of enzyme activity was defined as the amount of enzyme that produced 1 μmol reducing sugar per minute.
Morphology analysis
The system contained a piece of filter paper (1 cm × 2 cm) and 2.5 mg protein in 5 mL acetic acid–sodium acetate buffer (50 mM, pH 5.0), and the mixture was statically incubated at 55 ℃ for 24 h. After being treated with Pp-ArCel5, Pp-ArCel5-NG, Pp-ArCel5-LD, Pp-ArCel5-D and BSA, respectively, the morphology of the filter papers was observed.
Hydrature index analysis
The system contained 200 mg filter paper and 2.5 mg protein in 5 mL acetic acid–sodium acetate buffer (50 mM, pH 5.0), and the mixture was statically incubated at 55 ℃ for 24 h. After being treated with Pp-ArCel5, Pp-ArCel5-NG, Pp-ArCel5-LD, Pp-ArCel5-D and BSA, respectively, removed the enzyme solution on the surface of the filter paper, the filter paper was weighed as wet weight (Ww). Its dry weight (Wd) was obtained when the treated filter paper was dried at 60 ℃ to the constant weight after being washed three times with distilled water. The dried filter paper after treated with Pp-ArCel5, Pp-ArCel5-NG, Pp-ArCel5-LD, Pp-ArCel5-D and BSA was abbreviated as FPWT, FPNG, FPLD, FPCD and FPBSA, respectively. The hydrature index (HyI) (Lan et al. 2016) was calculated as (Eq. 1):
$${\text{HyI}}\% = \frac{{W_{{\text{w}}} - W_{{\text{d}}} }}{{W_{{\text{d}}} }} \times 100.$$
(1)
The dried filter paper was subjected to SEM, XRD, FTIR, and synergistic effect analysis separately.
XRD analysis
The crystalline index of FPWT, FPNG, FPLD, FPCD and FPBSA were analyzed by X-ray diffraction (XRD) using a Rigaku D/Max-3B diffractometer (Tokyo, Japan) with Cu Kα radiation (λ = 0.154 nm). 30 kV accelerating voltage and 30 mA current. The samples were detected in the range of 2θ between 5° and 50° with a scanning velocity of 2° min−1. The crystalline index (CrI) (Park et al. 2010) was calculated according to the following equation (Eq. 2):
$${\text{CrI }}(\% ) = (1 - {\text{ham}} / {\text{hcr}}) \times 100,$$
(2)
in which hcr is the peak height at 2θ = 22.5° and ham is the peak height at 2θ = 18°.
FTIR analysis
The Fourier transformed infrared spectroscopy (FTIR) spectral ranges were recorded from 400 to 4000 cm−1 using Spectrum One-B spectrometer (PerkinElmer, USA). 1 mg of FPWT, FPNG, FPLD, FPCD and FPBSA mixing with 100 mg of spectroscopic grade KBr, pressed into pellets and then placed into the plate that was cleaned with acetone twice. Spectrometer with the detector at 4 cm−1 and 100 scans per sample were signal-averaged and stored.
Synergistic action between Pp-ArCel5 mutants and cellulase
All reactions were done in 50 mM acetic acid–sodium acetate buffer, pH 5.0, at 55 °C with shaking 120 rpm over 24 h, and the total reaction volume was 200 μL. The final concentration of FPWT, FPNG, FPLD, FPCD, FPBSA and FP (untreated filter paper) used in the assay was 10 mg/mL, and Aspergillus niger cellulase (Sigma-Aldrich, St. Louis, USA) concentration was 20 µg/mg substrate. The FPBSA and FP treated by fungal cellulase were used as the control experiment.
Statistical analysis
All analyses were performed in triplicate, and all data were expressed as the means ± standard errors of the means. Data were subjected to one-way analysis of variance (ANOVA). A P-value < 0.05 was considered to be significant, following SPSS 11 (SPSS Inc., Chicago, IL, USA) software analysis.