Bacterial strains, plasmids, and culture condition
Paenibacillus agarexedens BCRC 17346 was purchased from BCRC (Bioresource Collection and Research Center, Hsinchu, Taiwan) and was used for the isolation of genomic DNA. Escherichia coli ECOS™ 9-5 (Yeastern, Taipei, Taiwan) was used for the propagation and manipulation of recombinant DNA. E. coli BL21(DE3) (Merck Millipore, Darmstadt, Germany) was used as the expression host. pJET1.2 (Fermentas, Maryland, USA) and pET-29a(+) (Merck Millipore) were used as cloning and expression vectors, respectively. P. agarexedens BCRC 17346 was cultured at 30 °C in nutrient broth medium supplemented with 0.1% (w/v) urea and 1% (w/v) glucose. E. coli was grown at 37 °C in Luria–Bertani (LB) medium (Difico, Detroit, USA) containing 30 μg/mL kanamycin, when required.
General DNA techniques
Bacterial genomic DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Plasmid DNA was isolated using the Plasmid Miniprep Purification Kit II (GMbiolab, Taichung, Taiwan) according to the manufacturer’s instructions. DNA fragments were amplified using GDP-HiFi DNA Polymerase (Genedirex, Las Vegas, USA) according to the manufacturer’s recommendations. All polymerase chain reactions (PCRs) were performed on a TProfessional TRIO thermocycler (Biometra GmbH, Göttingen, Germany). PCR products were purified using a PCR Clean-Up Kit (GMbiolab). Restriction enzyme digestions were performed according to the supplier’s recommendations (Thermo Fisher Scientific, Waltham, USA). DNA fragments were recovered from gels by using a Gel Elution Kit (GMbiolab). DNA ligation reactions were performed using a DNA Ligation Kit (Yeastern). The purified PCR product was cloned into pJET1.2 by using a CloneJET PCR Cloning Kit (Fermentas) according to the manufacturer’s recommendations. Plasmids were introduced into E. coli through heat shock transformation according to the manufacturer’s instructions.
Whole-genome sequencing of P. agarexedens
The genomic DNA of P. agarexedens was isolated and quantified using a Quant-iT dsDNA BR assay (Thermo Fisher Scientific). The quality of the extracted genomic DNA was verified on a 0.6% agarose gel. DNA libraries were constructed using an Illumina TruSeq DNA LT Sample Prep Kit. Mate-pair libraries were constructed using an Illumina Mate Pair Library Prep Kit v2. For the de novo assembly of the complete genome, we used Velvet (Zerbino and Birney 2008) to assemble paired-end and mate-pair reads to scaffolds. Genes on these assembled scaffolds were predicted using GeneMark.hmm (Besemer and Borodovsky 1999) and were annotated using a BLAST (blastp) search against the NCBI nr protein database, with an e-value cutoff of 0.00001. For the functional annotation of predicted genes, the accession numbers from BLAST hits were mapped to GO terms by querying the GO database (Ashburner et al. 2000). Enzyme code annotations were retrieved by mapping to GO terms, and enzyme codes were retrieved by querying the GO database.
Construction of expression vector
Based on the results of whole-genome sequencing of P. agarexedens, the forward primer PBAGA4F (5′-GATATAGGTACCGCCACGCCGTTCCCTACTC-3′, KpnI site underlined) and the reverse primer PBAGA4R (5′-CAATATCTCGAGTTAGTGGTGGTGGTGGTGGTGCTTTGAGATTAGCAGACGATCCATTA-3′; XhoI site underlined, stop codon in italics, and His tag DNA in bold) were designed and used to amplify the 2598-bp DNA fragment encoding the mature β-agarase AgaB-4 lacking the predicted signal peptide through PCR. This fragment was generated from the genomic DNA of P. agarexedens BCRC 17346. The PCR product was purified and then ligated to pJET1.2. The ligation mixture was transformed into E. coli ECOS™ 9-5 competent cells for the generation of recombinant plasmids. The recombinant plasmids were confirmed by DNA sequencing. The resulting plasmid containing the agarase DNA fragment was named pJET-AGAB-4. The agarase DNA fragment was excised from pJET1.2 by using KpnI and XhoI and was subsequently subcloned into pET-29a(+) at the corresponding restriction sites. The recombinant plasmid, designated pET-AgaB-4, was confirmed by DNA sequencing and was then transformed into E. coli BL21(DE3).
Expression and detection of rAgaB-4 in the soluble fraction
E. coli BL21(DE3)(pET-AgaB-4) cells were cultured in LB medium containing kanamycin (30 μg/mL), with shaking at 37 °C. On the next day, 0.1 mL of the overnight culture was inoculated into 10 mL of LB medium containing kanamycin (30 μg/mL) and was grown at various temperatures (37, 30, 25, 20, 16 °C), with shaking. When the optical density at 600 nm (OD600) of the cultures reached 0.4–0.6, isopropyl-β-d-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM was added. After 4 and 24-h incubation, cells were harvested by centrifugation at 10,000×g for 10 min at 4 °C and were then resuspended in lysis buffer (50 mM Tris–HCl and 500 mM NaCl, pH 8.0). Cells were lysed by sonication in an ice water bath. The suspensions (total cell lysate) were centrifuged at 10,000×g for 10 min at 4 °C. The clear supernatant (soluble fraction) was collected, and the remaining pellet (insoluble fraction) was resuspended in an equal volume of lysis buffer. Equal volumes of the total cell lysate, soluble fraction, and insoluble fraction were analyzed through 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970), using a minigel apparatus (model AE-6450; ATTO, Tokyo, Japan).
Western blot analysis
Protein samples were separated through 12.5% SDS-PAGE. After electrophoresis, proteins were electrophoretically transferred onto methanol-activated polyvinylidene fluoride membrane (Merck Millipore). The membrane was blocked with 5% skim milk in phosphate-buffered saline and incubated with the Penta·His antibody (1:20,000) (Qiagen), followed by incubation with an alkaline phosphatase-conjugated anti-mouse antibody (1:20,000) (Bethyl, Montgomery, USA). Immunoreactive bands were visualized using BCIP/NBT substrate solution (PerkinElmer, Waltham, USA).
Purification of rAgaB-4
E. coli BL21(DE3)(pET-AgaB-4) cells were cultured in 1 L of LB broth containing kanamycin (30 μg/mL), with shaking at 37 °C. Cells were cultured to an OD600 of 0.4–0.6. Subsequently, IPTG (0.1 mM) was added to induce rAgaB-4 expression at 20 °C for 24 h. Cells were harvested by centrifugation at 8000×g for 30 min and were then resuspended in lysis buffer. The cell suspension was disrupted using Constant Cell Disruption Systems (Constant Systems Ltd, Warwick, UK). The cell lysate was centrifuged at 8000×g for 15 min at 4 °C, and the resulting supernatant was filtered through a 0.22-μm membrane and applied to a 5-mL HiTrap™ excel affinity chromatography column (GE Healthcare, Uppsala, Sweden) according to the manufacture’s instruction. The purity of the eluted fusion protein was analyzed through 12.5% SDS-PAGE, and the protein concentration was determined using a Protein Quantification Assay Kit (MACHEREY–NAGEL, Düren, Germany).
Enzyme activity measurements
Agarase activity was measured by determining the amount of reducing sugars generated from hydrolysis, according to the DNS method developed by Miller (1959), with minor modifications. Briefly, 50 μL of suitably diluted rAgaB-4 solution was mixed with 950 μL of phosphate buffer (50 mM, pH 6) containing 0.2% (w/v) low-melting point (LMP) agarose. After incubation at 40 °C for 10 min, the sample was mixed with 1.0 mL of 3,5-dinitrosalicylic acid reagent solution, heated in a boiling water bath for 10 min, and then cooled in an ice water bath. Absorbance (OD) readings at 540 nm were obtained on an Infinite 200 PRO microplate reader (Tecan Group Ltd, Männedorf, Switzerland). The amount of enzyme required to produce 1 μmol d-galactose per min under the assay conditions was defined as one unit (U) of agarase. d-galactose was used as a reference reducing sugar for preparing the standard curve.
Effects of pH and temperature on agarase activity and stability
The effect of pH on rAgaB-4 activity was assayed at 40 °C in 50 mM buffer solutions containing 0.2% LMP agarose and 1.51 μg of purified rAgaB-4 with a pH range of 3–10 (at 1.0 intervals). The buffer solutions used were citric acid/sodium citrate buffer (pH 3–6), phosphate buffer (pH 6–8), and glycine–NaOH buffer (pH 9–10). The effect of temperature on rAgaB-4 activity was determined by monitoring agarase activity at temperatures ranging from 20 to 80 °C in 50 mM phosphate buffer (pH 6) containing 0.2% LMP agarose and 1.51 μg of purified rAgaB-4 for 10 min. The thermostability of rAgaB-4 was determined by measuring the residual enzyme activity after incubation at temperatures ranging from 20 to 80 °C in 50 mM phosphate buffer (pH 6) containing 0.2% LMP agarose and 1.51 μg of purified rAgaB-4 for 1 h.
Effect of various metal ions and ethylenediaminetetraacetic acid (EDTA) on enzyme activity
The effects of various metal ions and EDTA on rAgaB-4 activity were assayed in 50 mM sodium phosphate buffer (pH 6) containing 0.2% LMP agarose and 1.91 μg of purified rAgaB-4 by adding metal ions or EDTA at a final concentration of 1 mM. Hydrolysis reactions were performed at 55 °C for 10 min. Relative activity was calculated as the enzyme activity of rAgaB-4 with added metal ion or EDTA/activity of rAgaB-4 × 100.
Substrate specificity of rAgaB-4
The substrate specificity of rAgaB-4 was measured using high-melting point (HMP) agarose, LMP agarose, agar, sodium alginate, carrageenan, soluble starch, and sodium carboxymethyl cellulose. Hydrolysis reactions were performed at 55 °C for 10 min in 50 mM sodium phosphate buffer (pH 6) containing 0.2% substrates and 1.91 μg of purified rAgaB-4. Relative activity was defied as the percentage of activity determined with the respect to the maximum agarase activity.
Determination of kinetic parameters
The kinetic parameters of purified rAgaB-4 (1.54 μg) were determined in 50 mM phosphate buffer (pH 6) containing LMP agarose and HMP agarose (molecular mass, 120 kDa), ranging in concentration from 2 to 30 mg/mL. The reaction mixture was incubated at 55 °C for 10 min. Km and Vmax for LMP agarose and HMP agarose were determined from Lineweaver–Burk plots using SigmaPlot 12 software (Systat Software, San Jose, USA). Subsequently, the Kcat (turnover number) and Kcat/Km (catalytic efficiency) values were calculated based on the Vmax, Km, and [E] (concentration of rAgaB-4) values.
Thin layer chromatography analysis of hydrolysis products
The products of LMP agarose, HMP agarose, and agar hydrolysis by rAgaB-4 were detected using thin layer chromatography (TLC) performed on silica gel 60 plates (Merck Millipore) as previously described (Li et al. 2014) with some modifications. Hydrolysis reactions were conducted at 40 °C for 24 h in 50 mM sodium phosphate buffer (pH 6) containing 1% (w/v) LMP agarose, HMP agarose and agar with 1.54 μg of purified rAgaB-4, respectively. The reaction mixtures were centrifuged at 20,630×g for 10 min at 4 °C to pellet the undigested agarose and agar. Subsequently, 2 μL of each supernatant was applied to a silica gel 60 plate and was developed using an n-butanol-acetic acid-water solution (2:2:1, by volume). The developed oligosaccharides were detected by spraying the plate with aniline phthalate solution (Merck Millipore) and by heating it on a hot plate at 180 °C.
Evaluation of rAgaB-4 ability for DNA recovery from gel
The pUC19 plasmid (2.5 μg) was embedded in 1% LMP agarose. The agarose containing the pUC19 plasmid was incubated at 70 °C for 10 min and was treated with 1 U rAgaB-4 at 40 °C for 1 h. The mixture was centrifuged at 20,630×g for 10 min at 4 °C to remove the undigested residue. The DNA in the supernatant was precipitated by adding 0.6 volumes of isopropanol in the presence of 2.5 M ammonium acetate and 1 μg/μL glycogen. The mixture was centrifuged at 20,630×g for 10 min at 4 °C to pellet the DNA. The precipitated DNA was washed twice with 70% ethanol, dried, and dissolved in sterile Tris–HCl buffer (pH 8.0). Equal amounts of recovered DNA and the original pUC19 plasmid were analyzed through agarose gel electrophoresis.
Nucleotide sequence accession number
The nucleotide sequence of agaB-4 reported in this study has been submitted to the GenBank database under the accession number MF998080.