Dataset Information

Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate Lyase from Marine Bacterium Flammeovirga sp. NJ-04.

ABSTRACT: Alginate lyases have been widely used to prepare alginate oligosaccharides in food, agricultural, and medical industries. Therefore, discovering and characterizing novel alginate lyases with excellent properties has drawn increasing attention. Herein, a novel alginate lyase FsAlyPL6 of Polysaccharide Lyase (PL) 6 family is identified and biochemically characterized from Flammeovirga sp. NJ-04. It shows highest activity at 45 °C and could retain 50% of activity after being incubated at 45 °C for 1 h. The Thin-Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analysis indicates that FsAlyPL6 endolytically degrades alginate polysaccharide into oligosaccharides ranging from monosaccharides to pentasaccharides. In addition, the action pattern of the enzyme is also elucidated and the result suggests that FsAlyPL6 could recognize tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of -1 and +3. The research provides extended insights into the substrate recognition and degradation pattern of PL6 alginate lyases, which may further expand the application of alginate lyases.

SUBMITTER: Li Q

PROVIDER: S-EPMC6627919 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate Lyase from Marine Bacterium <i>Flammeovirga</i> sp. NJ-04.

Li Qian Q Hu Fu F Zhu Benwei B Sun Yun Y Yao Zhong Z

Marine drugs 20190531 6

Alginate lyases have been widely used to prepare alginate oligosaccharides in food, agricultural, and medical industries. Therefore, discovering and characterizing novel alginate lyases with excellent properties has drawn increasing attention. Herein, a novel alginate lyase FsAlyPL6 of Polysaccharide Lyase (PL) 6 family is identified and biochemically characterized from <i>Flammeovirga</i> sp. NJ-04. It shows highest activity at 45 °C and could retain 50% of activity after being incubated at 45 ...[more]

PMID: 31159265

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Project description:Exo-type agarases release disaccharide units (3,6-anhydro-l-galactopyranose-?-1,3-d-galactose) from the agarose chain and, in combination with endo-type agarases, play important roles in the processive degradation of agarose. Several exo-agarases have been identified. However, their substrate-degrading patterns and corresponding mechanisms are still unclear because of a lack of proper technologies for sugar chain analysis. Herein, we report the novel properties of AgaO, a disaccharide-producing agarase identified from the genus Flammeovirga AgaO is a 705-amino-acid protein that is unique to strain MY04. It shares sequence identities of less than 40% with reported GH50 ?-agarases. Recombinant AgaO (rAgaO) yields disaccharides as the sole final product when degrading agarose and associated oligosaccharides. Its smallest substrate is a neoagarotetraose, and its disaccharide/agarose conversion ratio is 0.5. Using fluorescence labeling and two-stage mass spectrometry analysis, we demonstrate that the disaccharide products are neoagarobiose products instead of agarobiose products, as verified by (13)C nuclear magnetic resonance spectrum analysis. Therefore, we provide a useful oligosaccharide sequencing method to determine the patterns of enzyme cleavage of glycosidic bonds. Moreover, AgaO produces neoagarobiose products by gradually cleaving the units from the nonreducing end of fluorescently labeled sugar chains, and so our method represents a novel biochemical visualization of the exolytic pattern of an agarase. Various truncated AgaO proteins lost their disaccharide-producing capabilities, indicating a strict structure-function relationship for the whole enzyme. This study provides insights into the novel catalytic mechanism and enzymatic properties of an exo-type ?-agarase for the benefit of potential future applications.Exo-type agarases can degrade agarose to yield disaccharides almost exclusively, and therefore, they are important tools for disaccharide preparation. However, their enzymatic mechanisms and agarose degradation patterns are still unclear due to the lack of proper technologies for sugar chain analysis. In this study, AgaO was identified as an exo-type agarase of agarose-degrading Flammeovirga bacteria, representing a novel branch of glycoside hydrolase family 50. Using fluorescence labeling, high-performance liquid chromatography, and mass spectrum analysis technologies, we provide a useful oligosaccharide sequencing method to determine the patterns of enzyme cleavage of glycosidic bonds. We also demonstrate that AgaO produces neoagarobiose by gradually cleaving disaccharides from the nonreducing end of fluorescently labeled sugars. This study will benefit future enzyme applications and oligosaccharide studies.

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Dataset Information

Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate Lyase from Marine Bacterium Flammeovirga sp. NJ-04.

Publications

Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate Lyase from Marine Bacterium <i>Flammeovirga</i> sp. NJ-04.

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