Abstract
Alginate lyases are important tools to prepare oligosaccharides with various physiological activities by degrading alginate. Particularly, the bifunctional alginate lyase can efficiently hydrolyze the polysaccharide into oligosaccharides. Herein, we cloned and identified a novel bifunctional alginate lyase, AlyA, with a high activity and broad substrate specificity from bacterium Isoptericola halotolerans NJ-05 for oligosaccharides preparation. For further applications in industry, the enzyme has been characterized and its action mode has been also elucidated. It exhibited the highest activity (7984.82 U/mg) at pH 7.5 and 55 °C. Additionally, it possessed a broad substrate specificity, showing high activities towards not only polyM (polyβ-d-mannuronate) (7658.63 U/mg), but also polyG (poly α-l-guluronate) (8643.29 U/mg). Furthermore, the Km value of AlyA towards polyG (3.2 mM) was lower than that towards sodium alginate (5.6 mM) and polyM (6.7 mM). TLC (Thin Layer Chromatography) and ESI-MS (Electrospray Ionization Mass Spectrometry) were used to study the action mode of the enzyme, showing that it can hydrolyze the substrates in an endolytic manner to release a series of oligosaccharides such as disaccharide, trisaccharide, and tetrasaccharide. This study provided extended insights into the substrate recognition and degrading pattern of the alginate lyases, with a broad substrate specificity.
Highlights
Alginate is the major component of the cell wall of brown algae [1]
The 16S rRNA sequence of the strain was sequenced (GeneBank No MH390700) and according to the phylogenetic analysis of the 16S rRNA sequence, the strain was assigned to the genus Isoptericola and named Isoptericola halotolerans NJ-05 (Figure 1)
The gene AlyA was cloned from the genome of Isoptericola halotolerans NJ-05 and sequenced (GeneBank No MH390701)
Summary
Alginate is the major component of the cell wall of brown algae [1]. It is a linear anionic heteropolysaccharide comprising of two uronic acids, α-L-guluronic (G) and β-D-mannuronic acid (M) [2]. Because of the high viscosity, gelling properties, and versatile activities, alginate has been widely applied in food, chemical, and pharmaceutical industries [4,5,6]. The applications of this polysaccharide are still limited, and are subjected to the high molecular weight and poor solubility [7]. Alginate oligosaccharide (AOS) has attracted more and more attention, as it retains the physiological functions and activities of alginate, but possessed smaller molecule weights and good bioavailability [8]. Yang et al found that AOS can enhance the uptake of LDL (Low-Density Lipoprotein) by regulating the expression
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