Identification and enzymatic characterization of an endo-1,3-b-glucanase from XXXX .

@entity1473 @entity26538 @entity3793 @entity72959 @entity1735 @entity40145 @entity4051 @entity214 @entity3689 @entity413 @entity12799
UNASSIGNED: Euglena produces paramylon as a storage polysaccharide, and is thought to require b-1,3-glucan degrading enzymes to release and utilize the accumulated @entity1735 . To investigate b-1,3-glucan degradation in Euglena, endo-1,3-b-glucanases were partially purified from @entity4051 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 @entity3793 -tag at the @entity1473 terminus was heterologously produced by @entity12799 and purified by immobilized metal affinity chromatography. Purified EgCel17A had a molecular weight of about 40kDa by @entity214 -PAGE, which was identical to that deduced from its @entity3689 sequence. The enzyme showed hydrolytic activity towards b-1,3-glucans such as @entity26538 and paramylon. Maximum activity of @entity26538 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 @entity26538 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 @entity413 , @entity40145 and @entity72959 . EgCel17A effectively produced soluble b-1,3-glucans from alkaline-treated Euglena freeze-dried powder containing paramylon. Thus, EgCel17 is the first functional endo-1,3-b-glucanase to be identified from @entity4051 .
