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A novel endo-type β-agarase was cloned from an agar-degrading bacterium, Microbulbifer sp. Q7 (CGMCC No. 14061), that was isolated from sea cucumber gut. The agarase-encoding gene, ID2563, consisted of 1800 bp that encoded a 599-residue protein with a signal peptide of 19 amino acids. Sequence analysis suggested that the agarase belongs to the GH16 family. The agarase was expressed in Escherichia coli with a total activity of 4.99 U/mL in fermentation medium. The extracellular enzyme activity accounted for 65.73% of the total activity, which indicated that the agarase can be extracellularly secreted using the wild-type signal peptide from Microbulbifer sp. Q7. The agarase exhibited maximal activity at approximately 40 °C and pH 6.0. It was stable between pH 6.0 and pH 9.0, which was a much wider range than most of the reported agarases. The agarase was sensitive to some metal ions (Cu2+, Zn2+ and Fe3+), but was resistant to urea and SDS. The agarase hydrolyzed β-1,4-glycosidic linkages of agarose, primarily yielding neoagarotetraose and neoagarohexaose as the final products. These indicate that this recombinant agarase can be an effective tool for the preparing functional neoagaro-oligosaccharides.
Fig. 1. The phylogenetic tree of the recombinant agarase
Fig. 2. SDS-PAGE and zymogram analysis of purified recombinant agarase. a SDS-PAGE analysis of the purified recombinant agarase. Lane M: protein markers. Lane 1: purified agarase. Lane 2: extracellular protein components. b Native-PAGE analysis of the purified agarase. Lane 1: purified agarase protein stained with Coomassie brilliant blue R-250. Lane 2: zymogram of the purified agarase. The native-PAGE gel was overlaid onto a sheet containing 2% (w/v) agarose in Tris–HCl buffer (50 mM, pH 7.0), incubated for 30 min at 40 °C, and then incubated with Lugol’s iodine solution to visualize agarase activity
Fig. 3. Characterization of the recombinant agarase. a The effect of temperature on agarase activity. b Thermal stability of recombinant agarase. The agarase was pre-incubated at various temperatures, and remaining activity was measured at 42 °C. c The optimal pH and pH stability of agarase was measured in 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 (pH 10.0 and 11.0)
Fig. 4. Determination of molecular masses of the hydrolytic products by mass spectrometry
Fig. 5.
13C-NMR spectrum of the reaction products of the agarase. The upper formula is the structure of neoagarohexaose. Peak assignments are labeled according to the nomenclature defined in the upper formula. A and G refer to the 4-O-linked 3,6-anhydro-α-l-galactopyranose and 3-O-linked β-d-galactopyranose; r and nr denote the reducing and non-reducing end; α/β for anomer
Fig. 6. TLC analysis of the products of agarose hydrolysis by the agarase and determination of the molecular
masses of the final products. a TLC analysis of the products of agarose hydrolysis by the agarase in different time. Hydrolysis reactions were conducted at 40 °C in 20 mM Tris–HCl buffer pH 7.2 containing 1% agarase substrate. Samples were taken at the indicated incubation times and analyzed by TLC as described in “Materials and methods sectionâ€. Determination of molecular masses of the two spots (b The first agarose-oligosaccharide spots from the TLC plate and c The second agarose-oligosaccharide spots from the TLC plate) recovered from the TLC plate showed that the final products of the reaction were neoagarotetraose and neoagarohexaose
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