Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

Abstract

bAlginate lyases are enzymes that degrade alginate through ␤-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (k cat ) for alginate of 0.60 ؎ 0.02 s ؊1 , 3.7 ؎ 0.3 s ؊1 , 4.5 ؎ 0.5 s ؊1 , and 7.1 ؎ 0.2 s ؊1 , respectively. The K m values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ؎ 7 M, 22 ؎ 5 M, 60 ؎ 2 M, and 123 ؎ 6 M, respectively. AlyA and AlyB were found principally to cleave the ␤-1,4 bonds between ␤-D-mannuronate and ␣-L-guluronate and subunits; AlyD and AlyE were found to principally cleave the ␣-1,4 bonds involving ␣-L-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers. Alginate is an abundant polysaccharide found within the cell wall of brown seaweeds (1) and comprises approximately 40% of the dry weight of the plant (2). Alginate is a copolymer consisting of the 1,4-linked epimers ␣-L-guluronate (G) and ␤-Dmannuronate (M). The individual monomeric subunits are organized in short stretches of polyguluronate (polyG), polymannuronate (polyM), or alternating sequences of mannuronate and guluronate (polyMG) (3). Alginate recently has been considered as a source for bioenergy, as it offers several potential advantages over terrestrial biomass. In particular, the farming of algae does not impinge on arable land; hence, it avoids the conflict between food and energy (4, 5). Algae also display higher growth rates than terrestrial plants (6). Finally, algae lack crystalline cellulose and lignin (7,8), bypassing a key obstacle to biofuel production.Alginate lyases are enzymes that degrade alginate through the ␤-elimination of the glycosidic bond into smaller oligomers. These enzymes can be classified based on the specific dyad G-G (EC 4.2.2.11) (9, 10), M-M (EC 4.2.2.3) (11), and M-G/G-M (12) bonds that they cleave. Additionally, alginate lyases can have either endocleaving or exocleaving specificity, with the majority of alginate lyases having an endocleaving preference (3). Along with the substrate specificity, alginate lyases also are characterized by their structure, termed polysaccharide lyase (PL) families. A total of seven PL families have been identified: PL5, PL6, PL7, PL14, PL15, PL17, and PL18 (13, 14). The most prevalent of these families is PL7 (Carbohydrate Active Enzymes database; http://www .cazy.org). The PL7 domain contains a ␤-jelly roll that consists of ␤-sheets in an antiparallel, adjacent barrel forming a cleft (12). This cleft contains three adjacent ␤-sheets that contain the catalytic residues (15). PL7 contains enzymes...

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

“…1C). Notably, both AlyA and A1-II= could degrade M-as well as G-enriched blocks (22,46), while AlyA1 was G specific (25).…”

“…84378061) (Fig. 1B) (22). Furthermore, due to their extraordinary sequence properties, Aly5 of Flammeovirga sp.…”

“…Most of the elucidated enzymes are endo-type lyases that internally and randomly degrade alginate, producing a series of unsaturated oligosaccharides with different degrees of polymerization (19,22,23). Only a few of these enzymes have been reported as exo-type enzymes that gradually cut the polysaccharide, yielding predominantly monosaccharides as products (24)(25)(26)(27)(28)(29).…”

Multicellular behavior enables cooperation in microbial cell aggregates

“…We used this model to explore how the physiochemistry of aggregates regulates growth and efficiency of up-taking break down products in this geometry. We tested some of the predictions of our model using alginate-degrading isolates of the marine microbe Vibrio splendidus (12,(20)(21)(22)(23). Alginate-degrading microbes secrete different amounts of hydrolytic enzyme and have different requirements for cooperation, prompting us to ask how bacterial behavior in the population alters the structure of aggregates and the efficiency with which alginate is consumed by cells.…”

Purification and characterization of alginate lyase from locally isolated marinePseudomonas stutzeriMSEA04

“…The mechanism of alginate lyases is by degradation of alginate through β-elimination of the glycosidic bond, yielding various oligosaccharides [1]. These oligosaccharides have an unsaturated uronic acid at the non-reducing terminus and unsaturated uronic acid monomers in the non-reducing end [13], which is an unsatu-rated double bond between C4 and C5, showing strong absorption at 235 nm.…”