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

Author:
Takeda, Takumi, Nakano, Yuki, Takahashi, Machiko, Konno, Naotake, Sakamoto, Yuichi, Arashida, Ryo, Marukawa, Yuka, Yoshida, Eriko, Ishikawa, Takahiro, Suzuki, Kengo
Source:
Phytochemistry 2015 v.116 pp. 21-27
ISSN:
0031-9422
Subject:
Aspergillus oryzae, Euglena gracilis, affinity chromatography, amino acid sequences, anion exchange chromatography, complementary DNA, enzymes, freeze drying, gel chromatography, genes, glucose, glycosides, hydrophobicity, mass spectrometry, molecular weight, pH, polyacrylamide gel electrophoresis, polymerase chain reaction, polysaccharides, proteins
Abstract:
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.
Agid:
5402981