Main strains, plasmids, media, and primers
A. niger ZJUBE-1 was stored at the China Center for Type Culture Collection (Conservation No. CCTCC M2010132) and used for homologous expression of AnEGL. Agrobacterium tumefaciens AGL-1 was used for transformation of marine A. niger. Escherichia coli DH5α and Rosetta (DE3) (Tsingke, Beijing, China) were used for subcloning and heterologous expression of AnEGL and its mutants. Glucose peptide yeast (GPY) medium (40 g/L glucose, 20 g/L peptone, 5 g/L yeast extract, 4 g/L KH2PO4) was used for propagation of marine A. niger and constitutive expression of AnEGL. PDA medium (20 g/L potato powder, 20 g/L glucose, 20 g/L agar) was used for the generation and preservation of marine A. niger. The induced medium (IM) and minimal medium (MM) used for A. tumefaciens-mediated transformation (AMT) of marine A. niger were accordant with the protocol (Michielse et al. 2005). The plasmid pCAMBIA-hph-bgl1 was constructed in our previous study (Cai et al. 2019) and the primers used are shown in Additional file 2: Table S1.
Cloning of endoglucanase genes
About 107 spores of A. niger ZJUBE-1 were inoculated in 100 mL GPY medium in 250 mL shake flask and cultivated with shaking at 180 rpm at 30 °C for 3 d. Then the genomic DNA was extracted using DNAiso (Takara, Beijing, China) according to its protocol. The total RNA was extracted with the fungal total RNA isolation kit (Sangon, Shanghai, China) according to its protocol, and then the cDNA was obtained by reverse transcription using oligo(dT)16 primer. Subsequently, the EGL genes (egl1 and egl2) were amplified from genomic DNA and cDNA using primers, egl-F and egl-R. Finally, these two EGL genes were inserted into pUCm-T vector (Sangon, Shanghai, China) and the resulting plasmids pUCm-egla/eglb were checked by DNA sequencing using primers, M13F and M13R.
Construction of Mini-Ti vector
The skeleton of Mini-Ti vector was clone from pCAMBIA-hph-bgl1 using primers, vector-F and vector-R. The target gene egla was clone from pUCm-egla using primers, egla-F and egla-R. Then the target Mini-Ti vector pCAMBIA-hph-egla was constructed by ligating these two fragments using pEASY-Uni seamless cloning and assembly kit (TransGen, Beijing, China; this kit was used in the following ligation steps). The DNA fragment PgpdA-egla-Tcbh1-hph-PtrpC was checked by DNA sequencing using primers, sequence-F and sequence-R.
Transformation of marine A. niger
A. tumefaciens AGL-1 was transformed with pCAMBIA-hph-egla using freeze–thaw method (Wise et al. 2006). Standard procedures of AMT were used as described in the protocol (Michielse et al. 2005). Briefly, the fresh A. tumefaciens recombinant was inoculated in IM broth, cultivated until OD600 reached 0.6–0.8. About 100 μL induced A. tumefaciens recombinant and 106 spores of marine A. niger were mixed and spread on cellophane of IM agar, cultivated for 2 d at 23 °C. Then the cellophane as well as the strains was transferred onto MM agar with 200 μg/mL hygromycin B and 200 μg/mL cefotaxime sodium. Additional MM agar with equal concentration of antibiotics was poured onto the cellophane to enhance screening effect. This interlayer medium was cultivated at 30 °C until recombinants grew out. Positive recombinants were verified by PCR identification using primers, PgpdA-F and PgpdA-R. Single conidiospore isolation was performed for acquisition of homozygote as described in our previous study (Cai et al. 2019). The resulting recombinants were stored at 4 °C.
EGL activity was assayed using sodium carboxymethyl cellulose (CMC-Na) as substrate. The enzymatic reaction mixtures (500 µl) containing 50 µl enzyme solution and 450 µl CMC-Na solution (1% (w/v) CMC-Na, 0.1 M sodium citrate/citric acid, pH 4.0) were incubated for 5 min at 50 °C. The amount of reducing sugar released was determined by 3,5-dinitrosalicylic acid (DNS) assay (Wood and Bhat 1988). One unit of EGL activity was defined as the amount of enzyme required for release 1 µmol glucose equivalent per minute. The values in following text were determined by this method unless otherwise stated.
For constitutive expression of AnEGL, 107 spores of recombinant were inoculated in 250 mL GPY medium in 1000 mL shake flask, cultivated with shaking at 200 rpm at 30 °C for 6 d. After fermentation, the supernatant was obtained by filtration with two layers of gauze and concentrated about fivefold by ultrafiltration with molecular weight cut-off 10 kDa. Then the expression was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).
After constitutive expression, the mycelia were removed using two layers of gauze. Then the supernatant was concentrated and the saline ions as well as the impurities with small molecule were replaced by water using ultrafiltration (molecular weight cut-off 10 kDa). The pH of ultrafiltrate was adjusted to 7.0 with 0.1 M NaOH and then loaded on DEAE Sepharose column pre-equilibrated with 20 mM sodium phosphate buffer pH 7.0. The fractions were eluted with the gradient of 0–0.5 M NaCl at a flow rate of 1 mL/min. The purity of AnEGL in each eluent was determined by SDS-PAGE. Then the purified AnEGL was concentrated and the buffer was replaced by 20 mM sodium acetate buffer pH 4.0 by ultrafiltration (molecular weight cut-off 10 kDa). The resulting enzyme solution was stored at 4 °C.
The purified AnEGL was used for the study on enzymatic properties. In terms of pH, the enzymatic reaction mixtures (500 μL), including 50 μL properly diluted enzyme solution and 450 μL CMC-Na solution (1% (w/v) CMC-Na, 0.1 M sodium citrate/citric acid, pH 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, and 6.5), were incubated in water bath at 50 °C for 5 min. In terms of temperature, the enzymatic reaction mixtures (500 μL), including 50 μL properly diluted enzyme solution and 450 μL CMC-Na solution (1% (w/v) CMC-Na, 0.1 M sodium citrate/citric acid, pH 4.0), were incubated in water bath at different temperatures (20, 30, 40, 50, 60, 70, and 80 °C) for 5 min. In terms of metal ions (5 mM and 10 mM), the enzymatic reaction mixtures (500 μL), including 50 μL properly diluted enzyme solution, 5 μL or 10 μL metal ions solution (NaCl, Na2SO4, NaNO3, LiCl, AgNO3, MgCl2, CaCl2, NiCl2, CuSO4, ZnSO4, CrCl2, HgSO4, FeCl3, 0.5 M), and 445 μL or 440 μL CMC-Na solution (1% (w/v) CMC-Na, 0.1 M sodium citrate/citric acid, pH 4.0), were incubated in water bath at 50 °C for 5 min. In terms of salt concentration, the enzymatic reaction mixtures (500 μL), including 50 μL properly diluted enzyme solution and 450 μL CMC-Na solution (1% (w/v) CMC-Na, 0, 1.0, 2.0, 3.0, 4.0, 5.0 M NaCl, 0.1 M sodium citrate/citric acid, pH 4.0), were incubated in water bath at 50 °C for 5 min. In terms of thermostability, 50 μL properly diluted enzyme solution was added into 450 μL CMC-Na solution (1% (w/v) CMC-Na, 0.1 M sodium citrate/citric acid, pH 4.0) after incubated at 50, 60, 65, and 70 °C for 5, 10, 15, 20, 30, 40, 50, 60, 90, and 120 min. Then the enzymatic reaction mixtures were incubated in water bath at 50 °C for 5 min. In terms of the influence of salt concentration on thermostability, enzyme solution was diluted properly with 0.1 M sodium citrate/citric acid buffer pH 4.0 with different NaCl concentrations (0, 1.0, 2.0, 3.0, 4.0, 5.0 M), incubated at 65 °C for 4, 8, 12, 16, 20, 30, 60 min. Subsequently, 50 μL of the diluted enzyme solution was added into 450 μL CMC-Na solution (1% (w/v) CMC-Na, 0.1 M sodium citrate/citric acid, pH 4.0). Then the enzymatic reaction mixtures were incubated in water bath at 50 °C for 5 min. The highest EGL activity was defined as 100%.
The amino acid sequence of AnEGL was translated from the sequence of egl2, and then its signal peptide was predicted by SignalP (http://www.cbs.dtu.dk/services/SignalP-4.0/). The alignment of amino acid sequences with other solved EGLs was accomplished with DNAMAN 8. The homology modeling of AnEGL was accomplished with Discovery Studio 3.0 using the crystal structure of A. niger β-1,4-endoglucanase (EglA, PDB ID: 1KS4) as template (Khademi et al. 2002). The conserved substrate binding sites and catalytic residues were determined according to the solved structure of EglA. The MD simulation was performed with GROMACS at two temperatures (300 and 350 K) and three salt concentrations (0, 2, and 4 M NaCl). After simulation, the root mean square deviation (RMSD), root mean square fluctuation (RMSF), and salt bridges were calculated by GROMACS. The visualization of trajectories was accomplished by VMD.
Obtainment of mutant AnEGLs
In order to simplify protein expression and enzyme assay, the mutant AnEGLs as well as original AnEGL were expressed in E. coli Rosetta (DE3). Firstly, the plasmid pET-eglb used for expression of original AnEGL was constructed. Briefly, the eglb encoding mature peptide was amplified from pUCm-eglb using primers, eglb-F and eglb-R. The linearized pET28a was obtained by PCR using primers, pET-F and pET-R. The plasmid pET-eglb was constructed by ligating these two fragments. Secondly, the plasmids pET-mutant1/2/3/4/5/6/7 were constructed. In detail, the entire plasmid pET-eglb was amplified using seven pairs of primers (mutant1-F and mutant1-R, mutant2-F and mutant2-R, mutant3-F and mutant3-R, mutant4-F and mutant4-R, mutant5-F and mutant5-R, mutant6-F and mutant6-R, mutant7-F and mutant7-R), respectively. The amplified products were transformed into E. coli Rosetta(DE3) after digested with restriction endonuclease DpnI (Takara, Beijing, China). Thirdly, the plasmid pET-mutant8 was constructed. Briefly, four fragments of eglb were amplified from pET-eglb using primers, mutant8-1-F and mutant8-1-R, mutant8-2-F and mutant8-2-R, mutant8-3-F and mutant8-3-R, and mutant8-4-F and mutant8-4-R, respectively. The linearized pET28a was obtained by PCR using primers, pET8-F and pET8-R. The plasmid pET-mutant8 was constructed by ligating these five fragments. The eight recombinant plasmids were transformed into E. coli Rosetta(DE3). The positive mutant EGL genes were identified by DNA sequencing using primers, T7F and T7R.
As for protein expression, the recombinants were inoculated into 5 mL LB broth, cultivated with shaking at 200 rpm at 37 °C overnight. The culture was inoculated into 50 mL LB broth, cultivated at 37 °C 200 rpm until OD600 reached 0.4–0.6. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added into the culture to 0.1 mM of final concentration, cultivated with shaking at 200 rpm at 16 °C for 16 h. After expression, the crude enzyme solutions were obtained by cell disruption with ultrasonic wave. The influences of salt concentration and temperature on activity were determined as mentioned in “Material and methods” section of enzymatic properties.