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

Abstract

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...

A novel β-glucosidase from Saccharophagus degradans 2-40T for the efficient hydrolysis of laminarin from brown macroalgae

“…Metal ions including Mg 2+ , Ca 2+ , Mn 2+ , Ni 2+ , Cu 2+ , Fe 2+ , and Co 2+ act as cofactors or inhibitors of certain enzymes. There are several studies that Cu 2+ or Fe 2+ inhibits certain enzymes [ 8 ].…”

“…There have been many studies on laminarinases (i.e., β-1,3-glucanases) from a variety of marine organisms [ 13 – 15 ]. For example, Gly5M, which was reported as an endo-type β-glucanase from S. degradans 2-40 T , can efficiently hydrolyze laminarin into various oligosaccharides with a small amount of glucose [ 8 ]. However, these reported laminarinases including Gly5M did not show laminaribiase activity.…”

“…This high exo-type β-1,3-glucanase activity of Bgl1B toward laminarin is important for the saccharification of brown macroalgal biomass because the enzyme cost for saccharifying biomass accounts for a large part in producing biofuels and bio-based products [ 18 ]. Regarding the hydrolysis of laminarin, most studies have focused on the hydrolysis of laminarin into oligosaccharides by endo- and/or exo-β-glucanases (i.e., laminarinases) [ 8 – 10 ], but there has been no report on laminarin depolymerization using β-glucosidases (i.e., laminaribiases). Therefore, Bgl1B has great potential in industrial production of biofuels and bio-based products from brown macroalgal biomass.…”

“…Although there have been many studies on the utilization of brown macroalgae as feedstocks for the production of bio-based chemicals [ 5 – 7 ], most of them are related to depolymerization of alginate, not laminarin. In some cases of laminarin depolymerization, the studies explored β-1,3-glucanases that produce mainly oligosaccharides and a small amount of glucose simultaneously [ 8 – 10 ]. However, there have been no studies on β-glucosidases that catalyze the production of glucose as the sole product.…”

Update on Marine Carbohydrate Hydrolyzing Enzymes: Biotechnological Applications

“…This enzyme also showed transglycosylation activity toward β-1,3-oligosaccharides when laminarioligosaccharides were used as 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 [ 68 ].…”