Strains, plasmids and media
Microbulbifer sp. Q7 (CGMCC no. 14061) was isolated from the guts of sea cucumbers and cultured in 2216E medium, the whole genome of Q7 was sequenced using a HiSeq PE150 (Novogene Bioinformatics Technology Co. Ltd., China) (Yang et al. 2017). The E. coli DH5α were used for cloning, and the E. coli BL21(DE3) were used for protein expression. Both strain were cultured in Luria–Bertani (LB) medium containing 100 μg/mL ampicillin. The pProEX-HTa vector (Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Science) was used as the cloning and expression vector.
Sequence analysis of the ID2563 gene
Our previous work sequenced the Microbulbifer sp. Q7 genome and identified the agarase gene, ID2563 (Yang et al. 2017). The ID2563 sequence was deposited into NCBI under the Accession Number WP_066965750.1.DNAMAN software was used to analyze the sequence of the gene. The signalP 4.1 server (http://www.cbs.dtu.dk/services/SignalP/) was used to predict the signal peptide sequence of ID2563. Analysis of its physicochemical properties was performed using ProtParam (http://web.expasy.org/protparam/). The conserved domain and catalytic site were predicted by the Conserved Domain Database (https://www.ncbi.nlm.nih.gov/cdd/). Mega5.0 was used to construct a phylogenetic tree using the neighbor-joining method.
Cloning and expression of the recombinant agarase
Genomic DNA from Microbulbifer sp. Q7, was extracted using a total DNA extraction kit (Sangon, Shanghai China). For extracellular agarase expression, the ID2563 gene with its original signal sequence was amplified by using the following primers, ID2563-F (CGGGATCCATGAAAACCACTCAGGGCG, BamHI site underlined) and ID2563-R (CCCAAGCTTTTAATTACTTAGCACGAACTTATCC, HindIII site underlined). The amplicon was cloned into pProEX-HTa. The recombinant plasmid was transformed into E. coli DH5α and plated on LB supplemented with 100 μg/mL ampicillin. Successful cloning of ID2563 into pProEX-HTa was confirmed by sequencing. pProEX-HTa-ID2563 was transformed into E. coli BL21 (DE3) and plated on LB supplemented with 100 μg/mL ampicillin. For agarase expression, the E. coli BL21(DE3) containing pProEX-HTa-ID2563 were grown at 37 °C in LB medium supplemented with 100 μg/mL ampicillin. When the OD600 reached 0.6–0.8, isopropyl-β-thiogalactoside (IPTG) was added to a final concentration of 1 mM. Cell were incubated at 23 °C 160 rpm for 24 h.
Assay of enzyme activity
Agarase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method (Miller 1959). Briefly, 100 μL of enzyme was added to 900 μL of 20 mM Tris–HCl pH 7.2 containing 0.2% (w/v) agarose, and the reaction was incubated at 40 °C for 5 min. One milliliter of DNS reagent added, and the reaction was heated in boiling water for 5 min and rapidly cooled. The absorbance was measured at 520 nm and compared with a standard curve for d-galactose. Enzyme activity (1 U) was defined as the amount of enzyme required to liberate 1 μM of d-galactose per min.
Purification of recombinant agarase
BL21(DE3) E. coli expressing his-tagged agarase were pelleted by centrifugation (10,000 rpm, 10 min). The supernatant and pellet were used to determine extracellular and intracellular agarase activity, respectively. Pelleted cells were resuspended in phosphate buffer saline and lysed by ultrasonication. Cell debris was removed by centrifugation (12,000 rpm, 10 min). Extracellular his-tagged agarase was purified using a Ni Sepharose 6FF column (GE Healthcare, USA) and imidazole concentrations between 10 and 400 mM. Fractions that were positive for agarase activity were pooled and concentrated using an ultrafiltration concentrator. Purified agarase was detected by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Agarase concentration was measured using the prestained protein ladder (Thermo, range 10–180 kDa).
Native-PAGE and zymogram analysis
Native-PAGE of the purified recombinant agarase solution was performed on 10% gel at 4 °C. Zymogram analysis gel was soaked in Tris–HCl buffer (50 mM, pH 7.0) for 5 min after the native-PAGE. Then the gel was overlaid onto a sheet of 2% (w/v) agarose in Tris–HCl buffer (50 mM, pH 7.0) and incubated at 40 °C for 30 min. To visualize agarase activity, the agarose sheet was flooded with Lugol’s iodine solution. Then the gel was removed from the agarose sheet and stained with Coomassie Brilliant Blue R-250.
Properties of enzyme
Agarase activity was measured at six temperatures between 30 and 60 °C under the standard conditions to determine the optimum temperature for activity. The thermal stability of agarase was determined based on its enzymatic activity after pre-incubation at different temperatures.
The optimum pH for agarase was determined by assessing its activity at different pH values. Three buffers was used: 50 mM Na2HPO4-citric acid (pH 3.0, 4.0 and 5.0), 50 mM sodium phosphate (pH 6.0, 7.0 and 8.0), 50 mM Tris–HCl (pH 9.0) and 50 mM Na2CO3-NaOH buffer (pH 10.0 and 11.0). Extracellular agarase was pre-incubation in the buffers listed above for 2 h at 20 °C and activity was measured to determine pH-dependent stability.
To determine the effects of ions and other molecules on agarase activity, the assay was performed in the presence of the following reagents: Na+ and Fe2+ (5, 20 and 50 mM), K+, Mg2+, Ca2+, Li+, Fe3+, Zn2+, Cu2+, EDTA, SDS, DTT and Urea (5 mM), 0.5% (v/v) ethylene thioglycol, 0.5% (v/v) Tween-80 and 0.5% (v/v) Triton-100. Enzyme activity was measured at 42 °C and pH 7.0. Reactions in the absence of the additives were used as controls.
Analysis of enzymatic hydrolytic products
The appropriate concentration of agarase was incubated with 1% agarose at 37 °C for approximately 6 h. Then solution was separated from the undegraded agarose by centrifugation. Macromolecular agarose and impurities were precipitated from the supernatant using different ratios of alcohol to supernatant (the maximum ratio is 6:1). The end products, the supernatant of the maximum ratio precipitation, were freeze-dried (FD-1A-50 vacuum freezer dryer, Xi An DP Biological Technology, China) for use in further experiments.
The molecular masses of the end products were detected by using electrospray ionization mass spectrometry (ESI–MS). Agar-oligosaccharide samples were dissolved in acetonitrile/1 mM NH4HCO3 (1:1, v/v) and analyzed with the micromass Q-TOF and Q-TOF ultima instruments (Waters, Manchester, UK) in negative-ion mode.
The end products were also analyzed by 13C-NMR (carbon-13 nuclear magnetic resonance) spectroscopy. The lyophilized powder was dissolved in D2O and spectra were recorded on an Agilent ProPulse 500 MHz NMR system. MestRe Nova software was used to analyze the 13C-NMR results. Deuterated acetone was used as an internal standard.
Agarase was added to 30 mL of 20 mM Tris–HCl pH 7.2 containing 1% (w/v) agarose. The reaction were incubated at 40 °C for 0–5 h and quenched by boiling for 5 min to denature the agarase. Subsequently, the reaction products were applied to a thin layer chromatography (TLC) silica gel 60 F254 plate (Merck, Darmstadt, Germany) using a solution of n-butyl alcohol/acetic acid/distilled water (2:1:1, v/v/v) as the mobile phase. The plate was sprayed with 10% H2SO4 in alcohol and heated to 110 °C for 10 min to visualize the product spots. To determine the composition of the final oligosaccharide products, the first two spots on the TLC plate were removed. Spots were dissolved in a small volume of distilled water and analyzed by ESI–MS (Agilent Technologies 6460 Triple Quad LC/MS) to determine their molecular weights.