Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2–40

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bAutotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) of Escherichia coli for the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes from Saccharophagus degradans 2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 ؋ 10 4 molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing E. coli.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes by Escherichia coli with a Novel Ligation-Independent Cloning Vector Based on the Autotransporter YfaL

Park

Song

et al. 2012

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“…Most agarases are endo-type enzymes that randomly and internally degrade agarose, producing a series of even-numbered oligosaccharides as products (16), and a few, mainly GH50 ␤-agarases, are exo-type enzymes that yield neoagarobiose (NA2) as the sole final agarose degradation product (22,23). In bacterial agarose degradation, endo-type agarases have an important role in initially depolymerizing agarose into oligomers, while exo-type agarases are essential for releasing the disaccharide units to facilitate subsequent sugar metabolism in bacteria (24,25).…”

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confidence: 99%

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

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...

“…In addition to these microorganisms, the marine bacterium Saccharophagus de-gradans 2-40 T , which is known as a superdegrader in the marine environment, utilizes at least 10 complex polysaccharides (18). Many enzymes for depolymerization of complex polysaccharides, including cellulases, xylanases, agarases, and alginate lyases, were characterized in S. degradans T (19)(20)(21)(22)(23)(24). However, enzymes depolymerizing laminarin in S. degradans 2-40 T have not been identified and characterized.…”

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confidence: 99%

A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40 ^T and Its Transglycosylase Activity

Wang

Kim

Seo

et al. 2016

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In this study, we characterized Gly5M, originating from a marine bacterium, as a novel ␤-1,3-1,6-endoglucanase in glycoside hydrolase family 5 (GH5) in the Carbohydrate-Active enZyme database. The gly5M gene encodes Gly5M, a newly characterized enzyme from GH5 subfamily 47 (GH5_47) in Saccharophagus degradans 2-40 T . The gly5M gene was cloned and overexpressed in Escherichia coli. Through analysis of the enzymatic reaction products by thin-layer chromatography, high-performance liquid chromatography, and matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry, Gly5M was identified as a novel ␤-1,3-endoglucanase (EC 3.2.1.39) and bacterial ␤-1,6-glucanase (EC 3.2.1.75) in GH5. The ␤-1,3-endoglucanase and ␤-1,6-endoglucanase activities were detected by using laminarin (a ␤-1,3-glucan with ␤-1,6-glycosidic linkages derived from brown macroalgae) and pustulan (a ␤-1,6-glucan derived from fungal cell walls) as the substrates, respectively. This enzyme also showed transglycosylase activity toward ␤-1,3-oligosaccharides when laminarioligosaccharides were used as the substrates. Since laminarin is the major form of glucan storage in brown macroalgae, Gly5M could be used to produce glucose and laminarioligosaccharides, using brown macroalgae, for industrial purposes. IMPORTANCEIn this study, we have discovered a novel ␤-1,3-1,6-endoglucanase with a unique transglycosylase activity, namely, Gly5M, from a marine bacterium, Saccharophagus degradans 2-40 T . Gly5M was identified as the newly found ␤-1,3-endoglucanase and bacterial ␤-1,6-glucanase in GH5. Gly5M is capable of cleaving glycosidic linkages of both ␤-1,3-glucans and ␤-1,6-glucans. Gly5M also possesses a transglycosylase activity toward ␤-1,3-oligosacchrides. Due to the broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value ␤-1,3-and/or ␤-1,6-oligosaccharides. Marine macroalgae have been considered as potential sources of carbohydrates for the production of biofuels and biologically based products (1). Among the marine macroalgae, over 70 million tons of brown macroalgae are produced annually (2). The major carbon storage compounds of brown algae are alginate, laminarin, fucoidan, and mannitol (3). Laminarin is a linear polysaccharide composed of D-glucose with ␤-1,3-glycosidic linkages in the main backbone and degrees of polymerization (DPs) of 20 to 25 (4), with some 6-O-branching from D-glucose in the main chain and some ␤-1,6-glycosidic linkages as branches; the ␤-1,3-glucan/␤-1,6-glucan ratio is approximately 3:1, depending on the species of brown macroalgae (5). Among brown macroalgae, the main sources of laminarin are Laminaria and Saccharina spp. For instance, Laminaria hyperborean and Saccharina latissima contain laminarin at up to 32% of dry weight (6), and 84% of total carbohydrates are laminarin in Fucus vesiculosus (7). Unlike other marine polysaccharides, such as alginate and agar, from which monomeric sugars are not fermentable by terrestrial microorganisms (8, 9), the final hydrolys...