Purification and characterization of an extracellular (1 --> 6)-beta-glucanase from the filamentous fungus Acremonium persicinum.
An endo-(1 --> 6)-beta-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 --> 6)-beta-glucans (pustulan and lutean), initially yielding a series of (1 --> 6)-beta-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as beta-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent bydrolysis of (1 --> 6)-beta- and some (1 --> 3)-beta-linkages in this substrate. K(m) values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Reagent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.
Pitson
S M
SM
Biotechnology Research Centre, La Trobe University Bendigo, Victoria, Australia.
Seviour
R J
RJ
McDougall
B M
BM
Stone
B A
BA
Sadek
M
M
D000164:Acremonium / Q000201:enzymology* / Q000254:growth & development; D002413:Cations, Divalent / Q000494:pharmacology; D002473:Cell Wall / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D013379:Substrate Specificity
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D004024:Dicyclohexylcarbodiimide; D006026:Glycoside Hydrolases; C020105:endo-1,6-beta-glucanase