Identification and enzymatic characterization of an endo-1,3-β-glucanase from Euglena gracilis

• Published in: Phytochemistry (Oxford) 2015-08, Vol.116, p.21-27
• Main Authors: Takeda, Takumi, Nakano, Yuki, Takahashi, Machiko, Konno, Naotake, Sakamoto, Yuichi, Arashida, Ryo, Marukawa, Yuka, Yoshida, Eriko, Ishikawa, Takahiro, Suzuki, Kengo
• Format: Article
• Language: English
• Subjects: beta-Glucans - analysis; Electrophoresis, Polyacrylamide Gel; Endo-1,3-β-glucanase; Euglena; Euglena gracilis - chemistry; Euglena gracilis - enzymology; Euglena gracilis - genetics; Euglenaceae; Glucan Endo-1,3-beta-D-Glucosidase - isolation & purification; Glucan Endo-1,3-beta-D-Glucosidase - metabolism; Glycoside hydrolase family 17; Glycoside Hydrolases - metabolism; Hydrolysis; Molecular Weight; Paramylon; Transglycosylation
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2015.05.010

An Euglena endo-1,3-β-glucanase, EgCel17A, catalyzed hydrolytic and transglycosylation reactions of β-1,3-linked oligosaccharides [a degree of polymerization greater than 3] and polymer; however, ultimately the substrates were degraded into glucose, laminaribiose and laminaritriose. [Display omitted] •Three endo-1,3-β-glucanases were partially purified from Euglena gracilis and their genes were identified.•Recombinant enzyme, EgCel17A, catalyzed hydrolytic and transglycosylation reactions.•EgCel17A functioned to degrade β-1,3-glucans to glucose, laminaribiose and laminaritriose. Euglena produces paramylon as a storage polysaccharide, and is thought to require β-1,3-glucan degrading enzymes to release and utilize the accumulated carbohydrate. To investigate β-1,3-glucan degradation in Euglena, endo-1,3-β-glucanases were partially purified from Euglena gracilis by hydrophobic, gel filtration and anion-exchange chromatography. Tryptic digests and mass-spectrometric analysis identified three proteins in the purified fraction as a member of glycoside hydrolase family (GH) 17 and two members of GH81. These genes were cloned from an Euglena cDNA pool by PCR. EgCel17A fused with a histidine-tag at the carboxy terminus was heterologously produced by Aspergillus oryzae and purified by immobilized metal affinity chromatography. Purified EgCel17A had a molecular weight of about 40kDa by SDS–PAGE, which was identical to that deduced from its amino acid sequence. The enzyme showed hydrolytic activity towards β-1,3-glucans such as laminarin and paramylon. Maximum activity of laminarin degradation by EgCel17A was attained at pH 4.0–5.5 and 60°C after 1h incubation or 50°C after 20h incubation. The enzyme had a Km of 0.21mg/ml and a Vmax of 40.5units/mg protein for laminarin degradation at pH 5.0 and 50°C. Furthermore, EgCel17A catalyzed a transglycosylation reaction by which reaction products with a higher molecular weight than the supplied substrates were initially generated; however, ultimately the substrates were degraded into glucose, laminaribiose and laminaritriose. EgCel17A effectively produced soluble β-1,3-glucans from alkaline-treated Euglena freeze-dried powder containing paramylon. Thus, EgCel17 is the first functional endo-1,3-β-glucanase to be identified from E. gracilis.