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.
Highlights
Alginate is a major acidic polysaccharide in brown algae, such as Sargassum vulgare (Sari-Chmayssem et al, 2016), Ecklonia radiate (Lorbeer et al, 2015), and Turbinaria ornate (Zubia et al, 2008); it is found in the exopolysaccharide of several bacteria, including Pseudomonas aeruginosa (Mccaslin et al, 2015)
The degradation products of AlyM were analyzed by infrared spectroscopy (IR), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) spectroscopy, and electrospray ionization mass spectrometry (ESI-MS)
Escherichia coli BL21 (DE3) cells harboring HTa-alyM were cultivated in LB medium to produce recombinant alginate lyase
Summary
Alginate is a major acidic polysaccharide in brown algae, such as Sargassum vulgare (Sari-Chmayssem et al, 2016), Ecklonia radiate (Lorbeer et al, 2015), and Turbinaria ornate (Zubia et al, 2008); it is found in the exopolysaccharide of several bacteria, including Pseudomonas aeruginosa (Mccaslin et al, 2015). Alginate degradation products have been developed for broad applications in agricultural, health product and medical industries, since they exhibit biological activities including immunomodulation, anti-tumor, antioxidant, and plant growth-promoting activities (Yokose et al, 2009; Tusi et al, 2011; Zhou et al, 2015). Various alginate lyases have been cloned and characterized, such as AlyA1, AlyA2 and AlyA3 from Azotobacter vinelandii, alyA from Pseudoalteromonas atlantica AR06, AlyFRA and AlyFRB from Falsirhodobacter sp. It is of great importance to find new alginate lyases to meet with the industrial application. According to the sequencing result of the whole genome of Q7, five alginate lyase-encoding sequences were predicted (Yang et al, 2018). A gene alyM, encoding alginate lyase AlyM, was studied for the cloning and expression. The degradation products of AlyM were analyzed by infrared spectroscopy (IR), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) spectroscopy, and electrospray ionization mass spectrometry (ESI-MS)
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