Abstract
The neoagaro-oligosaccharides, degraded from agarose by agarases, are important natural substances with many bioactivities. In this study, a novel agarase gene, agaW1540, from the genome of a deep-sea bacterium Shewanella sp. WPAGA9, was expressed, and the recombinant AgaW1540 (rAgaW1540) displayed the maximum activity under the optimal pH and temperature of 7.0 and 35 °C, respectively. rAgaW1540 retained 85.4% of its maximum activity at 0 °C and retained more than 92% of its maximum activity at the temperature range of 20–40 °C and the pH range of 4.0–9.0, respectively, indicating its extensive working temperature and pH values. The activity of rAgaW1540 was dramatically suppressed by Cu2+ and Zn2+, whereas Fe2+ displayed an intensification of enzymatic activity. The Km and Vmax of rAgaW1540 for agarose degradation were 15.7 mg/mL and 23.4 U/mg, respectively. rAgaW1540 retained 94.7%, 97.9%, and 42.4% of its maximum activity after incubation at 20 °C, 25 °C, and 30 °C for 60 min, respectively. Thin-layer chromatography and ion chromatography analyses verified that rAgaW1540 is an endo-acting β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose as the main products. The wide variety of working conditions and stable activity at room temperatures make rAgaW1540an appropriate bio-tool for further industrial production of neoagaro-oligosaccharides.
Highlights
Agar is the main glycan component that is segregated from the cell wall of red algae, for instance, Gracilaria sp. and Gelidium sp. [1,2]
Agarases are divided into two types, owing to the different hydrolysis modes, i.e., α-agarase (EC 3.2.1.158), which acts on the α-1,3 glycoside bonds and yields AOSs with 3,6-anhydro-Lgalactose at the reducing ends of products, and β-agarase (EC 3.2.1.81), which cleaves the β-1,4 glycoside bonds to generate NAOS with β-D-galactose at the reducing ends of products [5,6]
In the phylogenetic trees constructed by the methods of maximum likelihood (Figure 1a), neighbor-joining (Figure 1b), and minimum evolution (Figure 1c), the nucleotide sequence of agaW1540 had the closest phylogenetic relationship with a β-agarase from Pseudoalteromonas sp
Summary
Agar is the main glycan component that is segregated from the cell wall of red algae, for instance, Gracilaria sp. and Gelidium sp. [1,2]. Agar is the main glycan component that is segregated from the cell wall of red algae, for instance, Gracilaria sp. As the major biopolymer and supporting structure in seaweed, a cocktail of agarose and agaropectin constitute the molecular structure of agar [3,4]. As the main component of agar, possesses a linear structure that is composed of the alternant subunit of β-D-galactose and 3,6-anhydroL-galactose [5]. Agarases that depolymerize agarose into agarooligosaccharides (AOS) or neoagarooligosaccharides (NAOS) have been found in several gram-negative bacteria isolated from submarine sediments, seaweeds, marine organisms, and seawater [2]. Agarases are divided into two types, owing to the different hydrolysis modes, i.e., α-agarase (EC 3.2.1.158), which acts on the α-1,3 glycoside bonds and yields AOSs with 3,6-anhydro-Lgalactose at the reducing ends of products, and β-agarase (EC 3.2.1.81), which cleaves the β-1,4 glycoside bonds to generate NAOS with β-D-galactose at the reducing ends of products [5,6].
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