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AMB Express
2017 Dec 19;71:220. doi: 10.1186/s13568-017-0525-8.
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Extracellular expression of a novel β-agarase from Microbulbifer sp. Q7, isolated from the gut of sea cucumber.
Su Q
,
Jin T
,
Yu Y
,
Yang M
,
Mou H
,
Li L
.
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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 section”. 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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