Abstract
Neoagarobiose (NA2) is the repeating disaccharide unit of agarose and possesses various promising biological activities. To identify an efficient exolytic β-agarase required for NA2 production from agarose, the GH50A β-agarase gene from agar-degrading Cellvibrio sp. KY-GH-1 was overexpressed as a recombinant His-tagged protein using the Escherichia coli expression system. GH50A β-agarase that consists of 797 amino acids was able to produce predominantly NA2 from agarose at an optimal temperature and pH of 35 °C and 7.5, respectively. The enzyme was stable up to 35 °C and within a pH range of 7.0–9.0. The Km, Vmax, Kcat, and Kcat/Km values of the enzyme were 26.5 mg/mL, 16.9 U/mg, 25.2 s–1, and 1.2 × 105 s–1 M–1, respectively. The copresence of 5 mM MnSO4 and 10 mM tris(2-carboxyethyl)phosphine (TCEP) resulted in a 2.5-fold enhancement of the enzyme activity. For NA2 production, neoagaro-oligosaccharides (NAOSs) containing NA4–NA18 were preferred over agarose or agaro-oligosaccharides (AOSs) as substrates. NA2 was produced along with minor amounts of agarotriose (A3) after treatment of AOS with the enzyme, indicating that the exolytic digestion of AOS by the enzyme was initiated by releasing A3 from nonreducing ends. Enzymatic hydrolysis of 0.4% agarose (100 mL) using GH50A β-agarase (20 μg/mL) for 4 h under optimal reaction conditions (5 mM MnSO4, 10 mM TCEP, 35 °C, 20 mM Tris–HCl, and pH 7.5) and purification of NA2 from hydrolysis products by Bio-Gel P-2 column chromatography resulted in the recovery of 216 mg of NA2 (∼54% yield from agarose). Altogether, these results suggest that the recombinant GH50A β-agarase is useful to convert agarose to NA2.
Highlights
Agar, a well-known marine red algae cell wall polysaccharide, consists of a mixture of two different components: neutral agarose and negatively charged agaropectin
Comparing amino acid sequence similarities among bacterial agarases, the Carbohydrate-Active Enzymes (CAZyme) database classifies agarases into different glycoside hydrolase (GH) families: GH16, GH50, GH86, and GH118 for β-agarases,[7−9] as well as GH96 and GH117 for α-agarases.[10,11]
We examined which recombinant His-tagged βagarases expressed in the E. coli had the most prominent exolytic activity toward agar degradation
Summary
A well-known marine red algae cell wall polysaccharide, consists of a mixture of two different components: neutral agarose and negatively charged agaropectin. KY-GH-1 genome demonstrated the presence of three GH50 β-agarase genes (GH50A, GH50B, and GH50C).[27] Based on these data, we examined which recombinant His-tagged βagarases expressed in the E. coli had the most prominent exolytic activity toward agar degradation. TLC analysis data revealed that GH50A β-agarase could degrade an AOS mixture with various DPs to produce NA2 as the major product This process produced minor amounts of A3 that was presumably generated at the first step of the enzyme-catalyzed digestion of AOS (Figure 6C). LC-MS/MS analysis of the AOS enzyme-catalyzed hydrolysates showed that the major product NA2 and the minor product A3 were produced in a time-dependent manner (Figure 8) These results demonstrate that the AOS exolytic digestion by GH50A βagarase to produce NA2 is initiated after releasing A3 from the nonreducing end of AOS. Lyophilization of fractions 21−23 resulted in recovery of NA2 powder (184 mg), indicating ∼61% yield of NA2 from AOS
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