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

Abstract

In this study, we characterized Gly5M, originating from a marine bacterium, as a novel β-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 β-1,3-endoglucanase (EC 3.2.1.39) and bacterial β-1,6-glucanase (EC 3.2.1.75) in GH5. The β-1,3-endoglucanase and β-1,6-endoglucanase activities were detected by using laminarin (a β-1,3-glucan with β-1,6-glycosidic linkages derived from brown macroalgae) and pustulan (a β-1,6-glucan derived from fungal cell walls) as the substrates, respectively. This enzyme also showed transglycosylase activity toward β-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.

Importance: In this study, we have discovered a novel β-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 β-1,3-endoglucanase and bacterial β-1,6-glucanase in GH5. Gly5M is capable of cleaving glycosidic linkages of both β-1,3-glucans and β-1,6-glucans. Gly5M also possesses a transglycosylase activity toward β-1,3-oligosacchrides. Due to the broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value β-1,3- and/or β-1,6-oligosaccharides.

FIG 1

Effects of pH on Gly5M activity. Enzymatic reactions were performed for 30 min at 40°C in 20 mM glycine-HCl (pH 2.0 to 4.0), sodium acetate (pH 4.0 to 6.0), Tris-HCl (pH 6.0 to 9.0), or glycine-NaOH (pH 9.0 to 10.0).

FIG 2

Relative activity of Gly5M at different temperatures (A); relative activity of 30-min-preincubated Gly5M at different temperatures, measured to determine the thermal stability of Gly5M (B). Enzymatic activity was measured by reactions performed for 30 min at pH 6.0 and 40°C, using laminarin as the substrate.

FIG 3

TLC (A) and HPLC (B) analyses of Gly5M enzymatic reaction products using laminarin as the substrate. The reactions were performed at 30°C in 20 mM Tris-HCl (pH 6.0). Lane 1, glucose; lane 2, laminarin; lane 3, laminarin plus Gly5M for 5 min; lane 4, laminarin plus Gly5M for 30 min; lane 5, laminarin plus Gly5M for 60 min; lane 6, laminarin plus Gly5M for 120 min. The control contained substrate but lacked enzyme, and no incubation was performed.

FIG 4

TLC (A) and HPLC (B) analyses of Gly5M enzymatic reaction products using pustulan as the substrate. The reactions were performed at 30°C in 20 mM Tris-HCl (pH 6.0). Lane 1, glucose; lane 2, pustulan; lane 3, pustulan plus Gly5M for 5 min; lane 4, pustulan plus Gly5M for 30 min; lane 5, pustulan plus Gly5M for 60 min; lane 6, pustulan plus Gly5M for 120 min. The control contained substrate but lacked enzyme, and no incubation was performed.

FIG 5

MALDI–TOF/TOF MS analyses of Gly5M enzymatic reaction products using laminarin (A) and pustulan (B) as the substrates.

FIG 6

TLC (A) and MALDI–TOF/TOF MS (B) analyses of Gly5M enzymatic reaction products using laminarioligosaccharides as the substrates.

FIG 7

Phylogenetic analysis of Gly5M (GenBank accession no. ABD82280.1) and other characterized bacterial β-1,3-glucanases (EC 3.2.1.39) in the CAZy database. GH16 sequences (from Thermotoga maritima MSB8 [GenBank accession no. AAD35118.1], from Bacillus circulans [GenBank accession no. AAA22474.1], from Flavobacterium johnsoniae UW101 [GenBank accession no. ABQ07185.2], from Streptomyces sioyaensis [GenBank accession no. AAF31438.1], from Thermotoga petrophila RKU-1 [GenBank accession no. ABQ46917.1], from Nocardiopsis sp. F96 [GenBank accession no. BAE54302.1], from Paenibacillus sp. CCRC 17245 [GenBank accession no. ABJ15796.1], from Pseudomonas sp. PE2 [GenBank accession no. BAC16331.1], from Flavobacterium sp. 4221 [GenBank accession no. ABW02990.1], from Thermotoga neapolitana [GenBank accession no. CAA88008.1], from Streptomyces sp. S27 [GenBank accession no. ACO94508.1], from Bacillus circulans [GenBank accession no. AAC60453.1], from Cellulosimicrobium cellulans [GenBank accession no. AAC44371.1], and from Lysobacter enzymogenes [GenBank accession no. AAN77504.1]), GH64 sequences (from Cellulosimicrobium cellulans [GenBank accession no. AAA25520.1], from Arthrobacter sp. YCWD3 [GenBank accession no. BAA04892.1], from Streptomyces matensis [GenBank accession no. BAA34349.1], and from Lysobacter enzymogenes [GenBank accession no. AAN77503.1]), GH81 sequences (from Bacillus halodurans C-125 [GenBank accession no. BAB03955.1] and from Thermobifida fusca YX [GenBank accession no. AAZ56163.1]), and a GH55 sequence (from Arthrobacter sp. NHB-10 [GenBank accession no. BAF52916.1]) were aligned by using the UniProt Align tool (http://www.uniprot.org/align). The phylogenetic tree was inferred from the alignments with the minimum linkage method and was drawn by using MAFFT (version 7) (http://mafft.cbrc.jp/alignment/software).