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

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 40 kDa 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 1 h incubation or 50 °C after 20 h incubation. The enzyme had a K m of 0.21 mg/ml and a V max of 40.5 units/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 .