Purification and characterization of a cellulase from Dolabella auricularia.
A highly purified cellulase [EC 3.2.1.4] preparation (B beta) was obtained from a crude extract of gastric teeth of Dolabella auricularia by successive chromatographies on Sephadex G-100, DEAE-Toyopearl and CM-Toyopearl. The purified cellulase showed a single protein band on disc electrophoresis, and its isoelectric point was at pH 8.6. It contained relatively large amounts of basic amino acids and its molecular weight was estimated to be approximately 44,000 by sodium dodecyl sulfate (SDS) electrophoresis. The highest activity of this enzyme was attained at pH 6.3, but the enzyme was rather labile to heat. The activity of this enzyme was strongly inhibited by Hg2+, Mn2+, Zn2+, and Cu2+, whereas Ca2+ and Mg2+ showed no significant effect on the activity. The purified cellulase hydrolyzed sodium carboxymethyl cellulose (CMC) and phosphoric acid-swollen cellulose (swollen cellulose), as well as cellooligosaccharides and their reduction products, in an endowise fashion. It produced higher cellooligosaccharides effectively from swollen cellulose. Cellooligosaccharides with degrees of polymerization of 4-6 (G4-G6) were also hydrolyzed by the purified cellulase, but the modes of hydrolysis of these oligosaccharides were different from each other. The enzyme did not effectively attack cellooligosaccharides lower than G4. It produced G4 and G2 from G6, and G4 and glucose from G5. When these oligosaccharides were modified by reduction with sodium borotritide, the second linkage from the reducing end became, in each case, significantly susceptible to the enzyme and was preferentially cleaved.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002480:Cellulase / Q000302:isolation & purification* / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007525:Isoelectric Focusing; D008970:Molecular Weight; D008974:Mollusca / Q000201:enzymology*
D000596:Amino Acids; D002480:Cellulase