Cloning, expression and characterization of a cold-adapted endo-1, 4-β-glucanase from Citrobacter farmeri A1, a symbiotic bacterium of Reticulitermes labralis

Plasmids, chemicals, and culture medium

C. farmeri A1 that secreted cold-adapted EglC was isolated from R. labralis and stored in our laboratory. The plasmid pMD20-T vector (TaKaRa, Dalian, China) and E. coli DH5α were used for gene cloning, whereas the pET22b vector (Novagen, Madison, WI) and E. coli BL21 (laboratory stock) were used for gene expression. The flanking regions of the EglC gene were amplified using the TaKaRa Genome Walking Kit (TaKaRa, Dalian, China). Restriction endonucleases, T4 DNA ligase, and DNA polymerase were purchased from TaKaRa (Dalian, China). TIANgel Midi Purification Kit, TIANprep Mini Plasmid Kit, and 2 × Taq PCR Master Mix were purchased from Tiangen (Beijing, China).

EglC gene cloning

Genomic DNA of C farmeri A1 was isolated using the TIANamp Bacteria DNA Kit (Tiangen, Beijing, China). The core region of the EglC gene was amplified using the degenerate primers GH8-F and GH8-R (Table 1), which were designed based on two conserved blocks, EGQSY[A/G][M/L]FFAL and DAIRVY[L/M]WAG[M/L], of the glycoside hydrolase family 8 (GH8) bacterial endoglucanases. Touchdown PCR conditions were as follows: an initial denaturation step of 5 min at 95°C and 15 cycles of 30 s at 94 °C, 30 s at 70 °C, and 20 s at 72 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 55 °C, and 40 s at 72 °C, and a final elongation step of 10 min at 72 °C. Amplified DNAs of the appropriate size were purified and ligated into the pMD20-T vector, and confirmed by DNA sequencing (Genscript Corporation, Nanjing, China).

Full-length EglC was amplified by thermal asymmetric interlaced PCR (TAIL-PCR) using nested insertion-specific primers designed based on the conserved domain. The 5^′-flanking genomic sequences were amplified using FP-specific primers (FSP1, FSP2, and FSP3; Table 1) and the random degenerate primer C1, whereas the 3^′-flanking genomic sequences were amplified with RP-specific primers (RSP1, RSP2, and RSP3; Table 1) and the random degenerate primer C2 using the TaKaRa Genome Walking Kit (TaKaRa, Dalian, China). The TAIL-PCR products were ligated into the pMD20-T vector and transformed into E. coli DH5α for DNA sequencing. Then, the 5^′- and 3^′-flanking regions were assembled with the core sequence.

Based on the assembled sequence, the GHF and GHR primers (Table 1) were used to amplify the full length of the EglC gene. The PCR program included an initial step of 6 min at 95 °C, a second step of 35 cycles including 35 s at 95 °C, 1 min at 60 °C and 1 min at 72 °C, and a final step of 8 min at 72 °C The DNA product was cloned into the pMD20-T vector using standard procedures and sequenced by Genscript Corporation (China).

Construction and transformation of the recombinant expression vectors

For expression of EglC in E. coli, the mature protein-coding sequences (signal peptide excluded) were amplified by PCR from genomic DNA of C. farmeri A1. The PCR primers were EglC-F and EglC-R containing NcoI and XhoI sites (Table 1). The PCR program included an initial step of 6 min at 95 °C, a second step of 35 cycles including 35 s at 95 °C, 1 min at 58 °C and 1 min at 72 °C, and a final step of 8 min at 72 °C The purified PCR products were ligated into the pMD20-T plasmid. The vector pMD20-EglC was digested with NcoI and XhoI, and then the purified EglC gene was cloned to the pET22b vector. The resulting plasmid pET22b-EglC, was transformed into E. coli BL21 and plated on LB agar containing 100 µg ml⁻¹ ampicillin to select positive transformants. The positive clones were checked by colony PCR using EglC-F and EglC-R primers (Table 1). The plasmid pET22b-EglC was sequenced by Genscript Corporation (China).

Expression of the EglC gene in E. coli and purification of the recombinant protein

The recombinant E. coli pET22b-EglC was cultured at 37 °C in LB medium with ampicillin. When the culture density reached an optical density of approximately 0.5 at 600 nm, isopropyl-b-D-1-thiogalactopyranoside (IPTG) was added into the medium to a final concentration of 1 mM to induce endoglucanase expression. After 4 h, cultured cells were collected by centrifugation at 10,000 ×g for 6 min at 4 °C, resuspended in ice-cold buffer (Na₂HPO₄-citric acid; pH 7), and disrupted by sonication. Proteins were obtained by centrifugation and purified using a Ni²⁺ affinity chromatography column (CWBIO, China). Expressed proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and their concentration was determined using a Micro BCA protein assay kit (Jiancheng, Nanjing, China).

Detection of enzymatic activities

The activity of Eglc22b was determined using the 3,5-dinitrosalicylic (DNS) method as described by Fu et al. (2010). Sodium-carboxymethyl cellulose (CMC-Na) was used as a substrate at a concentration of 1.0% (w/v). The standard reaction mixture, containing 1 ml of appropriately diluted enzyme and 1 ml of CMC-Na buffer (pH 7), was incubated at 40 °C for 30 min. The reaction was terminated by addition of 3 ml of DNS to the mixture and 5 min of boiling. The release of reducing sugars was measured at 540 nm by using a spectrophotometer (Shanghai Precision & Scientific Instrument Co., Shanghai, China). One glucanase unit was defined as the amount of enzyme required to release 1 µmol of glucose per minute at the assay temperature.

Biochemical characteristics of EglC22b

The optimum pH for the activity of EglC22b was determined at 40 °C for 30 min in Na₂HPO₄-citric acid buffer (pH 3.5–7) and sodium phosphate buffer (pH 8.0). To determine the pH stability, EglC22b was incubated at 40 °C for 30 min in different buffers of pH 2.5 to 6.5 (1.0 interval), and the residual enzyme activity was measured at optimal pH and temperature using the DNS method as described previously.

The optimal temperature for the activity of EglC22b was determined in Na₂HPO₄-citric acid buffer (pH 7.0) at 10 °C to 70 °C (10 °C interval) for 30 min. To determine the thermal stability, EglC22b was incubated at 20, 30, 40, 50, and 60 °C for 30 min each, and the residual enzyme activity was measured at optimal pH and temperature using the DNS method as described previously.

The effects of different metal ions and chemical reagents on the activity of EglC22b were assessed by incubating the enzyme at 40 °C for 30 min in a standard reaction mixture of Na₂HPO₄-citric acid buffer (pH 7.0) containing CoCl₂ (2 mM), CdCl₂ (2 mM), ZnCl₂ (2 mM), FeCl₃ (2 mM), Li₂SO₄ (2 mM), Triton X-100 (10%), dimethyl sulfoxide (DMSO, 20%), acetonitrile (20%), Tween 80 (20%), SDS (0.4%), and ethylene-diamine-tetraacetic acid (EDTA, 20 mM). An assay system without the added metal ions was used as a control, and the enzyme activity was measured using the DNS method as described previously.

Statistical analysis

All measurements of the present study were carried out in duplicate. Data were analyzed by Microsoft Excel 2010. Data were presented as means with standard deviation (SD).

Cloning and sequencing of EglC

An approximately 600-bp core region of EglC was amplified by touchdown PCR from C. farmeri A1. Based on the sequence of the EglC core region, TAIL-PCR was used to amplify the flanking sequences of the DNA fragment. Finally, a 1,107 bp fragment was obtained from C farmeri A1 (Fig. 1). The open reading frame was predicted to encode a protein of 368 amino acids with a theoretical molecular mass of 39.1 kDa. Sequence analysis revealed a signal peptide with a length of 21 amino acids at the N-terminal of the enzyme. A BLAST search was used to predict the mature EglC of C. farmeri, revealing that the mature EglC belongs to glycoside hydrolase family 8 (Fig. 2). Additionally, the deduced EglC had 86.4%, 43.4%, 44.7%, 39.7%, and 44.3% amino acid sequence identity with endo-β-1,4-glucanases from E. coli CFT073, Burkholderia sp. CCGE1002, Cupriavidus taiwanensis, Pseudomonas fluorescens SBW25, and Xanthomonas campestris pv. vesicatoria strain 85-10, respectively (Fig. 3). The nucleotide sequence of EglC was deposited in the GenBank database (GenBank accession no. KT313000).

Expression and purification of EglC22b

The mature EglC gene without the signal peptide was cloned into the pET22b vector for expression. SDS-PAGE revealed that the apparent molecular mass of EglC22b was approximately 42 kDa (Fig. 4). This band was not present in non-transformed strains. The activity of the E. coli pET22b-EglC crude extract was 9.5 U/ml.

The crude EglC22b was purified using Ni²⁺-NTA affinity chromatography, as a protein with the expected MW (42 kDa) was present in the SDS-PAGE gel (Fig. 4, lane 5). The specific activity of the purified enzyme was 8.7 U/mg.

Effects of pH and temperature on the activity of EglC22b

The activity and stability of EglC22b were assayed in CMC-Na at different pH values and temperatures. EglC22b showed optimal activity at pH 7.0 and exhibited >94% and >85% relative activity at pH 6.5 and 8.0 (Fig. 5A). EglC22b was highly stable at pH 3.5–7.5 and retained >70 % residual activity after 30 min of incubation in these buffers (Fig. 5B).

The recombinant enzyme had an optimal temperature of 30–40 °C and exhibited >50% relative activity even at 5 °C (Fig. 6A). The activity of EglC22b was lost rapidly at temperatures higher than 60 °C (Fig. 6A). The thermotolerance analysis showed that approximately 90% of enzymatic activity was lost after 30 min of incubation at 60 °C (Fig. 6B)

Effects of chemical reagents and metal ions on the activity of EglC22b

The effects of various chemical reagents and metal ions on the activity of EglC22b were tested (Fig. 7). The presence of Cd²⁺, Zn²⁺, Li⁺, Triton X-100, DMSO, acetonitrile, Tween 80, EDTA, and SDS inhibited the activity of EglC22b. However, the presence of Co²⁺ and Fe³⁺ increased the activity of EglC22b.