Purification and characterizations of a novel recombinant Bacillus velezensis endoglucanase by aqueous two-phase system
© The Author(s) 2018
Received: 18 March 2018
Accepted: 11 April 2018
Published: 27 April 2018
Cellulases played an important role in the production of bioenergy and bio-products. Cellulases from bacteria with some special characteristics drew great attention due to its fast growth speed, wide adaption to harsh environment, and production of multi-function cellulases.
An endoglucanase gene egls from Bacillus velezensis A4 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant enzyme Egls was partially purified using aqueous two-phase system. The highest recovery rate of the enzyme was 90.39% at PEG 4000 (25% w/w), phosphate buffer 8.08% (w/w) (pH 6.0), and NaCl (5% w/w). The enzyme molecular weight was 55 KD estimated by zymogram. The optimal pH and temperature of recombinant enzyme Egls were pH 6.0 and 55 °C, respectively. The enzyme was stable at pH range of 5.0–7.0 at 55 °C for 60 min. The enzyme exhibited Km, Vmax, Kcat values as 63.38 mg/ml, 55.6 mg/min, and 3.93 × 103/S, respectively. The addition of 10 mM of Mg2+, Mn2+, or 5% (w/w) of Triton-X 100 in the reaction system enhanced the enzyme activity significantly. The enzyme showed both endoglucanase and exoglucanase activity.
Cellulose, hemicellulose, and lignin are the most abundant, cheap, and renewable nature resources in the world (Basu 2013). Cellulose accounts for about 1/3 of the lignocellulosic biomass (Sun and Cheng 2002). Hydrolysis of cellulose to produce reducing sugars using lignocellulosic biomass is extremely important in the production of renewable bioenergy and other bio-products (Pérez et al. 2002; Yang et al. 2014).
The biodegradation of cellulose was mainly conducted by various kinds of cellulase (Endoglucanase, exoglucanase, and β-glucosidase) (Garvey et al. 2013). Endoglucanases randomly cut the cellulose polysaccharide chain, generating various lengths of oligosaccharides. Exoglucanases act on the ends of cellulose polysaccharide chains, producing either glucose or cellobiose as major products. β-glucosidases hydrolyze cellobiose from non-reducing end to produce glucose (Rabinovich et al. 2002).
Cellulases could be produced by various fungi and bacteria. Fungi have the ability to produce abundant amounts of cellulolytic enzymes and are mainly exploited to produce cellulases (Sharada et al. 2013). However, cellulases from bacteria have been paid more attention because it has fast growth speed, wide adaption to extreme environment, and can produce more complex and muti-function cellulases to meet the industrial needs (Sadhu and Maiti 2013). Although numerous cellulases from different bacteria species have been studied and characterized (Rabinovich et al. 2002; Sadhu and Maiti 2013; Sharada et al. 2013), the cellulases with novel properties are still needed to be further explored for bioenergy production.
Aqueous two-phase systems (ATPS) are formed by mixing certain amounts of polymers, salts, and water together, which have been extensively used to separate and purify nuclear acids, cells and organelles, enzymes, and proteins (Asenjo and Andrews 2011; Raja et al. 2012). ATPS have many advantages such as low toxic to environment, continuous to operate, and easy to scale up (Hatti-kaul 2001). It is one of low cost and high-efficient purification methods (Asenjo and Andrews 2011). Recently, many enzymes (α-amylase, α-galactosidase, lipase, proteinase), recombinant proteins, and antibody have been purified using this method (Azevedo et al. 2009; Porfiri et al. 2011; Gu et al. 2012; Loc et al. 2013; Ramakrishnan et al. 2016).
Bacillus velezensis A4 was isolated from forest soil previously in our lab, which exhibited strong ability in hydrolysis of lignocellulosic biomass (Guo et al. 2017). In order to further investigate the lignocellulosic enzyme properties from the A4 strain, the endoglucanase encoding gene was cloned and expressed in Escherichia coli. The enzyme was purified with ATPS and enzyme properties were characterized.
Bacterial strain and cultural condition
The Bacillus sp. A4 strain was used in this study which was isolated from the forest soil (Guo et al. 2017). The strain was activated by streaking in Luria–Bertani (LB) plate incubating at 37 °C overnight. Then a single colony was inoculated to 20 ml of liquid medium and cultured overnight in a shaker at 37 °C with 200 rpm. One percent (v/v) of the cultural broth was transferred into 50 ml mineral salt medium (MSM) containing 0.05% (w/v) carboxymethylcellulose sodium (CMC) and cultured in a shaker at 37 °C with 200 rpm for 80 h. The MSM medium consisted of (g/l) KH2PO4 1.0 g, KCl 1.0 g, NaNO3 1.0 g, MnSO4 0.5 g, yeast extract 0.5 g, peptone 3 g, pH 7.0. The cultural medium was sterilized at 121 °C for 20 min before use.
rpo B gene amplification and strain identification
The genome DNA was extracted using the method (Aljanabi 1997). The rpoB gene fragment was amplified with the primers CM7: 5′-AACCAGTTCCGCGTTGGCCTGG-3 (1383 bp) and CM31b: 5′-CCTGAACAACACGCTCGGA-3 (2473 bp) (Mollet et al. 1997). The PCR-amplified fragment was purified with DNA purification kit (Thermo Fisher Scientific, Canada) and sent for sequencing. The rpoB gene sequence of the strain was blasted with GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The highly similar rpoB gene sequence from different Bacillus strains was retrieved from GenBank and aligned using software Clustal X 1.83. The phylogenetic tree was constructed with software MEGA 7.01 using the neighbor-joining method.
Egls gene cloning and sequence analysis
The endoglucanase gene (egls) fragment was amplified with the forward primer (5′-GGATCCATGCGAAGGAGGAAAAGATCAGAT) and reverse primer (5′-AAGCTTATTKGGTTCYGTTCCCCAAATCAGT). The restriction sites BamHI and HindIII were shown underline. The PCR product was purified with a DNA purification kit (Thermo Fisher Scientific, Canada). The fragment was ligated to the vector pJET1.2-T and transformed into E. coli JM109. The plasmid was extracted and digested with enzymes BamHI and HandIII. The egls fragment was purified with Gel Extraction kit and ligated with the vector pET-21a in the corresponding sites. The degenerated plasmid was named pET21-egls, which has a 6 His-tag at the C-terminus of egls gene. The plasmid was sent to sequence and analyze.
The Nucleotide and amino acid sequences were analyzed with the online software BLASTn and BLASTp (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The conserved domains were predicted using bioinformatics tools (http://www.ncbi.nig.gov/structure/cdd/wrpsb.cgi). The signal peptide of Egls was predicted using online program Signal P 4.2 (http://www.cbs.dtu.dk/services/SignalP/). The molecular weight (MW) and theoretical isoelectric point (PI) were analyzed using the software Vector NTI Advance 11.5.1. Multiple sequence alignment analysis was performed using software CLUSTAL × 1.8. Homology model was built using the online software (https://swissmodel.expasy.org/).
Recombinant protein expression
The recombinant strain E. coli DE3 harboring the plasmid pET21-egls was inoculated into LB broth supplemented with ampicillin 50 mg/l. The strain was grown at 37 °C with shaking at 200 rpm overnight, and then it was inoculated to the same medium and was grown to OD600 0.5–0.6. After that, the recombinant strain was induced with 1 mM IPTG at 20 °C for 20 h. The cell pellets were collected in 50-ml conical tubes by centrifuging at room temperature for 10 min at 5000 rpm. The enzyme was purified with HisPur™ Ni–NTA Spin Purification Kit (Thermo fisher, Canada) under the native conditions. The recombinant protein was eluted with 500 mM imidazole in phosphate buffer (pH 8.0) and desalted with PD-10 column (Thermo fisher, Canada). The protein concentration was assayed according to the instruction of Bradford Protein Assay Kit (Bio Basic Canada Inc., Markham, Canada). SDS-PAGE was performed with 5% (w/v) stacking gel and 12% (w/v) separating gel, respectively, under denature conditions. Zymogram analysis was carried out using the method (Lin et al. 2015).
Enzyme activity assay
Endoglucanase activity was assayed according to the method (Ghose 1987) with minor modification using CMC as substrate. The reaction was carried out by adding 50 μl enzyme extraction into 50 μl of 0.05% (w/v) CMC substrate in 50 mM citrate buffer (pH 4.8) and incubated at 55 °C for 30 min. The released reducing sugars were measured by DNS method (Miller 1959), using glucose as standard curve. One unit of enzyme activity was defined as the amount of enzyme which could release 1 μg of reducing sugar in 1 min under assay conditions.
Exoglucanase activity and filter paper activity (FPA) assay was carried out in the same conditions as endoglucanase determination using 1% (w/v) Avicel and Whiteman No. 1 filter paper (0.3 × 0.5 cm) as substrate, respectively. The released reducing sugars from Avicel were determined by phenol–sulfuric acid method (Masuko et al. 2005), using glucose as standard curve. One unit of enzyme activity was defined as the amount of enzyme which could release 1 μg of reducing sugars in 1 min under assay conditions.
The β-glucosidase activity was assayed according to the method (Zhang et al. 2009), with minor modifications using pNPG as substrate. Briefly, 10 μl of enzyme was added into 140 μl of 1 mM pNPG substrate in 50 mM sodium citrate buffer (pH 4.8) and incubated at 55 °C for 30 min. Then 200 μl of 0.4 M glycine buffer (pH 10.8) was added into the reaction mixture. The appearance of yellow color was monitored at 430 nm by Microplate Spectrophotometer (Epoch, Bio Tek Instruments, Inc., Winooski, VT, USA). One unit of enzyme activity was defined as the enzyme released 1 mM of p-nitrophenyl per minute under assay conditions.
Enzyme purification with ATPS
Binodal curves preparation
The bimodal curves were determined with turbidity method without NaCl (Asenjo and Andrews 2012). One g of 50% (w/w) PEG solution was added into a 15-ml tube. Then, the 40% (w/w) of citrate solution was added drop by drop, until the mixture becomes cloudy. The weight of PEG 4000 solution, phosphate buffer (pH 6.0), and the total weight of the tube were written down to calculate the ratio of PEG solution and citrate buffer solution in the system. After that some distilled water was added to make the system clear until one-phase system appeared. The above process was repeated 8–10 times until enough data were obtained to plot binodal curves.
Enzyme purification with two-phase system
Aqueous two-phase systems were prepared at room temperature by mixing certain amounts of 50% (w/w) of PEG solution, 40% (w/w) of phosphate buffer, 2 ml cell culture supernatant sonicated in 50 mM of citrated buffer (pH 6.0), and NaCl in 15 ml centrifuge tubes with conical cap. pH was pre-adjusted to (6.0–8.0) in phosphate buffer. Distilled water was added to the system to obtain 10 g of total weight. After vortex for 5 min, the tube was centrifuged at 5000 rpm for 3 min to make the phase separate. Different concentrations of PEG 4000 (10–25% w/w), phosphate buffer (8–18% w/w), buffer pH (6–8), and NaCl (0–15%, w/w) were used to study the partition of the recombinant enzyme in ATPS.
Partition coefficient (K) is the ratio of protein partitioned in the top phase to that of in the bottom phase. Purification factor (PF) is defined as the ratio of specific activity of the phase enzyme participated to that of the crude enzyme. Enzyme recovery (ER) is defined as the ratio of the total enzyme recovered from the phase to the total enzyme activity in crude enzyme.
Biochemical characterizations of Egls
The optimal pH and temperature
The optimal pH and temperature for endoglucanase activities were measured in different pH values (3.0–10.0) and various temperatures (30–75 °C), respectively, using 0.05% (w/v) of CMC in citrate buffer (pH 4.8) as substrates. Sodium acetate buffers (50 mM) were used to maintain the pH range of 3.0–5.0, phosphate buffer (50 mM) 6.0–8.0, and glycine–NaOH buffer (50 mM) 9.0–10.0, respectively. The enzyme activity compared to the highest activity in percentage (%) in the reaction conditions was shown as relative activity.
The thermal and pH stability
In thermal and pH stability assay, the purified Egls (50 μg/ml) was incubated at temperature range of 30–80 °C for 15–60 min in 50 mM citrate buffer (pH 4.8) at pH 3.0–10.0 for 5 h at room temperature. The remained enzyme activity was measured under standard conditions. The original enzyme activity was set as 100%. The relative enzyme activity (%) was defined as the remained enzyme activity compared with original enzyme activity.
Effects of metal ions and chemicals on endoglucanase on enzyme activity
Effects of metal ions and chemicals on Egls activities were determined using 2 and 10 mM of different metal irons (CaCl2, MnCl2, MgCl2, NiCl2, ZnCl2, CuCl2, and KCl) in 50 mM citrate buffer (pH 4.8) and chemicals (SDS, Triton-X 100, Tween-20, EDTA and PMSF), respectively. The enzyme activities were assayed under standard conditions. The enzyme activity without addition of metal ions was defined as 100% and used as a control. The results were shown as relative activity (%).
Kinetic parameters K m/k cat assay
Kinetic parameters Km, Vmax, and kcat were determined according to the procedure (Allison 2001). Twenty-two U/ml of enzyme was used to digest 1–10 mg/ml of CMC substrate under optimized conditions. The data were analyzed and the Lineweaver–Burk plot was made by the software Origin version 8.0.
Substrate specificity assay
Different substrates were used to evaluate the specificities of the purified enzyme Egls under the optimized conditions. The enzyme activity was measured under standard condition. The glucose was used as standard.
All the experiments were carried out in triplicate. The results were shown as mean ± standard deviation. One-way analysis of variance (ANOVA) was applied to analyze the significance of data. A p < 0.05 was considered as statistically significant in data using Tukey’s HSD method.
The gene sequence of egls and rpoB were submitted to the GenBank with the Accession No (MG748603 and MG748604), respectively.
Results and discussion
B. velezensis A4 growth and enzyme production
Egls gene sequence analysis
Computer model of enzyme Egls shows that it consists of a (α/β)8 barrel, 7 β sheet strands, and a Manganese(II) binding site (Fig. 4b). The proposed conserved domains have cellulase domain (residues 50–296) (glycosyl hydrolase family 5), a CBM_3 cellulose-binding domain (residues 356–437), and one Mg2+ binding site, related with amino acids (G157, D195, D197, N198). The enzyme showed 96.73% sequence identity with endoglucanase. Some strains of B. velezensis species have been sequenced and endoglucanases were annotated in GenBank by bioinformatics methods. However, the enzyme characterizations were not been clearly investigated.
Expression and purification of the recombinant enzyme
Purification of recombinant enzyme Egls using PEG 4000-phosphate-NaCl system
Total protein (μg/ml)
Total CMCase (U/ml)
Biochemical properties of Egls
The optimal pH and temperature
Endoglucanase properties from different Bacillus strains
Molecular weight (MW) (kDa)
Optimal temperature (°C)
B. velezensis A4
65 °C 30 min
75 °C 30 min
Li et al. (2008)
Lee et al. (2008)
Annamalai et al. (2012)
B. subtilis JS2004
Zafar et al. (2014)
The thermal and pH stability
The thermal stability was assayed at different temperatures (55–70 °C) for 1 h. The results showed that the enzyme was inactivated after incubation at 70 °C for 30 min (Fig. 7c, d). The crude enzyme and the purified enzyme were stable at the pH range of 5.0–7.0. Egls was not thermostable at temperatures above 70 °C and it was stable at nearly neutral pH environment. The enzyme properties are highly related to the structure of the enzyme. Some endoglucanases from Bacillus have higher thermostability, and others have a wider range of pH stability (Table 2).
Effects of metal ions and chemicals on endoglucanase on enzyme activity
Effect of metal irons and chemicals on purified recombinant enzyme Egls activities
Relative activity (%)
Relative activity (%)
100.3 ± 1.0
100.3 ± 1.0
100.3 ± 1.0
100.3 ± 1.0
105.2 ± 2.3
115.7 ± 5.2a
105.0 ± 4.4
122.9 ± 5.8a
105.5 ± 1.8
104.3 ± 4.6
100.8 ± 3.6
105.2 ± 2.5
111.0 ± 1.4a
134.8 ± 11.1a
99.6 ± 0.6
96.7 ± 0.5
101.7 ± 1.7
134.2 ± 4.9a
98.8 ± 3.1
98.6 ± 3.6
100.3 ± 3.4
122.0 ± 1.8a
100.0 ± 3.0
103.5 ± 4.8
110.1 ± 1.1a
116.5 ± 3.8a
Kinetic parameters K m/K cat
The kinetic parameters Km/Kcat were determined using various concentrations of CMC as substrate and analyzed with Lineweaver–Burk plot. The Km, Vmax, and Kcat values of Egls were exhibited as 63.38 mg/ml, 55.6 mg/min, and 3.93 × 103/S, respectively. The value Km/Kcat of Egls was calculated as 1.74 × 10−2 mg/ml S. The kinetic parameters (Km, Vmax, and Kcat) of an endoglucanase from Bacillus strain were found as 0.25 mg/ml, 20 μmol/ml/min, and 0.55/S respectively (Sadhu et al. 2013). The Km of endoglucanase from the strain Hahella chejuensis KCTC 2396 was 1.8 mg/ml (Ghatge et al. 2014). The higher Km value of our strain indicated that the enzyme Egls is less efficient in digesting CMC substrate than reported strains.
Substrates specificity of crude and purified recombinant enzyme Egls
Crude enzyme (U/ml)
Purified enzyme (U/ml)
22.06 ± 4.61
75.61 ± 8.45*
40.84 ± 6.38
69.22 ± 8.52*
63.86 ± 16.10
68.84 ± 24.81*
We have cloned and expressed egls gene from B. velezensis A4 in E. coli BL21 (DE3). The recombinant enzyme was purified with PEG 4000, phosphate, and NaCl aqueous two-phase system. Although gene sequence was highly similar to other B. velezensis stain JS25R, this study is the first report of the recombinant expression, purification, and characterization of the enzyme. The recombinant enzyme is an endoglucanase, has an optimal temperature at 55 °C and pH range of 5.0–6.0, is stable at pH range of 5.0–7.0, and is actively supplemented with a series mental ions and can be used in biomass pretreatment to produce reducing sugars in bioenergy production. The study of the enzyme properties of the predicted cellulase genes is very important for the enzyme application in biomass degradation and bioenergy production.
YL conceived, designed, and performed the experiments. HG constructed the E. coli expression vector. YW performed a part of the experiments. WQ was involved in project planning, experimental designing, manuscript revisions, and editing. All authors read and approved the final manuscript.
We acknowledge the financial support of Natural Sciences and Engineering Research Council of Canada (NSERC Grant No. RGPIN-2017-05366) and BioFuelNet Canada (Project No. 67) to W.Q and China Scholarship Council (Grant No. 201608330437) to Y. L. We thank Dr. Frooq Khan and Prof. Z. Jiang for the help and providing some experimental materials and conditions in this research.
The authors declare that they have no competing interests.
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