Expression and characterization of a thermostable and pH-stable β-agarase encoded by a new gene from Flammeovirga pacifica WPAGA1

Hou, Yanping; Chen, Xinglin; Chan, Zhuhua; Zeng, Runying

doi:10.1016/j.procbio.2015.04.005

Cited by 39 publications

(39 citation statements)

References 42 publications

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“…Because of the excellent agarose degradation and utilization capabilities of Flammeovirga strains, various agarase preparations, such as crude extracellular enzymes (29) and pure recombinant agarases (27,31,32), have been investigated. And all of these enzyme preparations yielded NA4 rather than NA2 as the smallest oligosaccharide product.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the two final oligosaccharide products appeared to be neoagarohexaose (NA6) and NA4 rather than NA4 and NA2 because the R f value of D-galactose was smaller than that of NA2 but larger than that of NA4 under the designated thin-layer chromatography (TLC) system (22,23,33,34). Therefore, all the three studied agarases of Flammeovirga strains produce NA4 as the smallest oligosaccharide product (31,32). However, the tetraoligosaccharide cannot be utilized directly and efficiently by bacteria until it is further enzymatically digested.…”

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confidence: 98%

“…strain MY04 could liquefy and grow on agarose as the sole carbon source, indicating that MY04 can produce efficient agarose-degrading enzymes (29). To date, one GH86 ␤-agarase (AgaP4383 of Flammeovirga pacifica strain WPAGA1) (31) and two GH16 ␤-agarases (AgaG4 of Flammeovirga sp. strain MY04 and AgaYT of Flammeovirga yaeyamensis strain YT) (27,32) have been identified from Flammeovirga strains.…”

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confidence: 99%

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Biochemical Characteristics and Substrate Degradation Pattern of a Novel Exo-Type β-Agarase from the Polysaccharide-Degrading Marine Bacterium Flammeovirga sp. Strain MY04

Han

Cheng

Wang

et al. 2016

Appl Environ Microbiol

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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. IMPORTANCEExo-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. Agarose is a complex polysaccharide that contains repeated disaccharide units of 3,6-anhydro-L-galactopyranose-␣-1,3-Dgalactose (1). Agarose has been identified to be one of the polysaccharide components of the cell wall and intercellular substances in red algae (Rhodophy...

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Section: Discussionmentioning

confidence: 99%

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confidence: 98%

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confidence: 99%

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Biochemical Characteristics and Substrate Degradation Pattern of a Novel Exo-Type β-Agarase from the Polysaccharide-Degrading Marine Bacterium Flammeovirga sp. Strain MY04

Han

Cheng

Wang

et al. 2016

Appl Environ Microbiol

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“…Although the bacteria of the genus Flammeovirga are able to digest complex polysaccharides, majority of the studies have focused on their ability to hydrolyse agar [27][28][29]. Here, for the first time, we report the characterization of an ␣-amylase (Amy16) from F. pacific, which we had isolated from deep-sea sediments of the west Pacific Ocean [15].…”

Section: Discussionmentioning

confidence: 99%

Characterization of a thermostable and alkali-stable α-amylase from deep-sea bacterium Flammeovirga pacifica

Zhou

Jin

Cai

et al. 2015

International Journal of Biological Macromolecules

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“…Most of these Flammeovirga strains can efficiently digest various polysaccharides, such as agarose, alginate, and starch, indicating that the strains contain abundant enzyme resources related to carbohydrate metabolism. Thus far, three ␤-agarases and one ␣-amylase have been identified in three different Flammeovirga strains (35)(36)(37)(38), although little is known about other types of polysaccharide depolymerases, such as alginate lyases. Due to the excellent polysaccharide-degrading capabilities of bacteria, the draft genome sequences of Flammeovirga sp.…”

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confidence: 99%

Novel Alginate Lyase (Aly5) from a Polysaccharide-Degrading Marine Bacterium, Flammeovirga sp. Strain MY04: Effects of Module Truncation on Biochemical Characteristics, Alginate Degradation Patterns, and Oligosaccharide-Yielding Properties

Han

Cheng

et al. 2016

Appl Environ Microbiol

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c Alginate lyases are important tools for oligosaccharide preparation, medical treatment, and energy bioconversion. Numerous alginate lyases have been elucidated. However, relatively little is known about their substrate degradation patterns and productyielding properties, which is a limit to wider enzymatic applications and further enzyme improvements. Herein, we report the characterization and module truncation of Aly5, the first alginate lyase obtained from the polysaccharide-degrading bacterium Flammeovirga. Aly5 is a 566-amino-acid protein and belongs to a novel branch of the polysaccharide lyase 7 (PL7) superfamily. The protein rAly5 is an endolytic enzyme of alginate and associated oligosaccharides. It prefers guluronate (G) to mannuronate (M). Its smallest substrate is an unsaturated pentasaccharide, and its minimum product is an unsaturated disaccharide. The final alginate digests contain unsaturated oligosaccharides that generally range from disaccharides to heptasaccharides, with the tetrasaccharide fraction constituting the highest mass concentration. The disaccharide products are identified as ⌬G units. While interestingly, the tri-and tetrasaccharide fractions each contain higher proportions of ⌬G to ⌬M ends, the larger final products contain only ⌬M ends, which constitute a novel oligosaccharide-yielding property of guluronate lyases. The deletion of the noncatalytic region of Aly5 does not alter its M/G preference but significantly decreases the enzymatic activity and enzyme stability. Notably, the truncated protein accumulates large final oligosaccharide products but yields fewer small final products than Aly5, which are codetermined by its M/G preference to and size enlargement of degradable oligosaccharides. This study provides novel enzymatic properties and catalytic mechanisms of a guluronate lyase for potential uses and improvements.A lginate is a linear polysaccharide composed of alternating residues of ␤-D-mannuronic acid (M) and its C-5 epimer, ␣-Lguluronic acid (G) (1). The uronic acid residues are arranged into homopolyuronic blocks of M residues (M block), G residues (G block), or heteropolyuronic blocks (MG or GM blocks). Alginate has been identified as a cell wall component of seaweeds belonging to Phaeophyta, such as kelp and sargassum (2, 3). Due to its ability to form a strong gel after absorbing water, algal alginate has been widely used as a supporting material in food, medical, and industrial applications (4, 5). Alginates containing acetyl modifications at the O-2 or O-3 positions have been purified from the extracellular matrix of some bacteria, such as the pathogen Pseudomonas aeruginosa and Azotobacter soil bacteria (6, 7). Understanding how to prevent Pseudomonas pathogens from synthesizing and secreting extracellular high-molecular-weight alginates is important for clinical therapy protocols (8, 9).Alginate lyase can break the 1 to 4 O linkages between the uronic acid residues of alginate via a ␤-elimination mechanism. The reaction forms C-4ϭC-5 double bonds within the s...

show abstract

Expression and characterization of a thermostable and pH-stable β-agarase encoded by a new gene from Flammeovirga pacifica WPAGA1

Cited by 39 publications

References 42 publications

Biochemical Characteristics and Substrate Degradation Pattern of a Novel Exo-Type β-Agarase from the Polysaccharide-Degrading Marine Bacterium Flammeovirga sp. Strain MY04

Biochemical Characteristics and Substrate Degradation Pattern of a Novel Exo-Type β-Agarase from the Polysaccharide-Degrading Marine Bacterium Flammeovirga sp. Strain MY04

Characterization of a thermostable and alkali-stable α-amylase from deep-sea bacterium Flammeovirga pacifica

Novel Alginate Lyase (Aly5) from a Polysaccharide-Degrading Marine Bacterium, Flammeovirga sp. Strain MY04: Effects of Module Truncation on Biochemical Characteristics, Alginate Degradation Patterns, and Oligosaccharide-Yielding Properties

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