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Expression and Characterization of a New PolyG-Specific Alginate Lyase From Marine Bacterium Microbulbifer Sp. Q7

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Expression and Characterization of a New PolyG-Specific Alginate Lyase From Marine Bacterium Microbulbifer Sp. Q7

Min Yang et al. Front Microbiol.

Abstract

Alginate lyases play an important role in preparation of alginate oligosaccharides. Although a large number of alginate lyases have been characterized, reports on directional preparation of alginate oligosaccharides by alginate lyases are still rather less. Here, a gene alyM encoding a new alginate lyase AlyM was cloned from Microbulbifer sp. Q7 and expressed in Escherichia coli. AlyM exhibited the maximumactivity at pH 7.0 and 55°C and showed special preference to poly-guluronic acid (polyG). Glycine promoted the extracellular secretion of AlyM by 3.6 times. PBS and glycerol significantly improved the thermal stability of AlyM, the enzyme activity remained 75 and 78% after heat-treatment at 45°C for 2 h, respectively. ESI-MS analysis suggested that AlyM mainly produced oligosaccharides with degrees of polymerization (DP) of 2-5. The results of 1H-NMR showed that guluronic acid (G) occupied the reducing end of the end products, indicating that AlyM preferred to degrade the glycosidic bond at the G-X linkage. HPLC analysis showed that the hydrolysis products with a lower degree of polymerization contained more G. Therefore, AlyM shows good potential to produce alginate oligosaccharides with specific M/G ratio and molecular weights.

Keywords: Microbulbifer; alginate lyase; extracellular secretion; heterologous expression; poly-guluronic acid preference; thermal stability.

Figures

Figure 1
Figure 1
(A) Nucleotide sequences and deduced amino acid sequences of the alginate lyase gene from Microbulbifer sp. Q7. The black box, red box (Q477-I478-H479), and asterisk mark the signal peptide sequence, catalytic residues and stop codon, respectively. The deduced amino acid sequence contains three conserved domains: CBM_4_9 (N17-G82), F5_F8_type_C (D177-M289), and alginate_lyase2 (F344-H609) domains. (B) Phylogenetic analysis of AlyM. This phylogenetic tree was calculated by the method of maximum likelihood. Genbank accession numbers and organism names are given. Branch numbers indicate the bootstrap values in the ML analysis. Arcs with different colors indicate different PL families.
Figure 2
Figure 2
Induction of IPTG (A) and lactose (B) on the extracellular expression of AlyM. The BL21-HTa-alyM was pre-incubated at 37°C for 2 h, then inducer with different concentrations (IPTG: 0.3, 0.5, 0.7, 0.9, 1.1, and 1.3 mM; lactose: 0, 0.4, 0.8, 1.2, and 1.6%) was added into the culture medium and cultivation continued at 23°C (IPTG) or 28°C (lactose) to determine the optimal inducer concentration. The BL21-HTa-alyM was pre-incubated at 37°C for 2 h, then 0.9 mM IPTG or 0.4% lactose was added into the culture medium and cultivated at different temperatures (IPTG:17, 20, 23, 26, and 29°C; lactose: 26, 28, 30, and 32°C) to determine the optimal induction temperature.
Figure 3
Figure 3
Effects of additives on the expression of AlyM. The additives included 0.5% (w/v) Gly, 0.02% (w/v) SDS, 0.2% (v/v) TritonX-100, and 2% (v/v) Tween-80. BL21-HTa-alyM was pre-incubated at 37°C for 2 h, then 0.4% lactose was added into the culture medium and cultivation continued at 28°C for 22 h.
Figure 4
Figure 4
SDS-PAGE analysis of recombinant AlyM expression and purification. M, marker (Thermo Fisher Scientific Inc., Waltham, MA, USA); 1, extracellular recombinant AlyM; 2, affinity-purified recombinant AlyM.
Figure 5
Figure 5
Biochemical characterizations of AlyM. (A) The optimal reaction temperature of the enzyme. (B) The optimal reaction pH of the enzyme. (C) Thermal stability of AlyM at pH 7.0. The purified enzyme was heat-treated at 35–50°C for 6 h and assayed for residual enzyme activity to evaluate the thermal stability. (D) The effect of NaCl, glycerol, and PEG1000 with different concentrations on AlyM thermal stability. The enzyme was heat-treated at 45°C for 2 h and the residual enzyme activity was measured to evaluate the thermal stability. (E) The effect of different buffers (PBS, Tris-HCl, acetate buffer, and citrate buffer) with different concentrations (50, 100, and 200 mM) on thermal stability of the enzyme. The enzyme was heat-treated in different buffer solutions at 45°C for 2 h and assayed for residual enzyme activity to evaluate the thermal stability. (F) Effects of chemical reagents on the enzyme activity. Reaction systems without addition of chemical reagents were used as control.
Figure 6
Figure 6
The CD spectrum of AlyM. The concentration of AlyM in 20 mM phosphate buffer (pH7.4) was set at 0.5 mg/mL, the 20 mM phosphate buffer was used as the blank.
Figure 7
Figure 7
ESI-MS of the AlyM hydrolysis products.
Figure 8
Figure 8
13C-NMR (A) and 1H-NMR (B) of AlyM hydrolysis products.
Figure 9
Figure 9
1H-NMR analysis of the end products of the digestion of alginate by AlyM. The fractions UDP2, UDP3, UDP4, and UDP5 were purified by Bio-Gel P4 Polyacrylamide Gel. “udp2” was the typical chemical shift of alginate oligosaccharide with UDP2.
Figure 10
Figure 10
IR analyses of alginate and the AlyM hydrolysis products. (a), alginate; (b), hydrolysis products.
Figure 11
Figure 11
HPLC analyses of hydrolysis products precipitated by ethanol with different volumes.

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