ABSTRACT
The time course production of xylanolytic enzymes by the rumen anaerobic fungus Neocallimastix frontalis was studied during growth on different carbon sources and revealed using isoelectric focusing and immunoblotting. A constant low level of endoxylanase expression was observed in glucose medium. A high level of xylanase activity was detected in methyl glucoside medium corresponding to the induction of new isoforms which were repressed by the presence of glucose. beta-Xylosidases were constitutively produced at a high level and remained mainly associated to the fungal cells. Polyclonal antibodies raised against the endoxylanases XYLI and XYLII revealed that XYLI was secreted to the different culture media showing a characteristic pattern of constitutive expression, while anti-XYLII recognized several polypeptides larger than XYLII indicating the production of multiple antigenically related enzymes during growth on the inducing substrate.
Subject(s)
Fungal Proteins/analysis , Fungi/enzymology , Rumen/microbiology , Xylosidases/analysis , Animals , Antibodies, Fungal , Fungal Proteins/metabolism , Glucose/pharmacology , Glucosylceramides/pharmacology , Methylglucosides/pharmacology , Rabbits , Sheep , Xylan Endo-1,3-beta-Xylosidase , Xylosidases/metabolismABSTRACT
beta-D-Glucosidase, beta-D-fucosidase, beta-D-xylosidase, and beta-D-cellobiopyranosidase activities in Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata, located with fluorescent conjugates, occur throughout the whole thallus as from zoospore germination and disappear before sporulation. beta-D-Galactosidase and alpha-L-arabinopyranosidase activities are low or nonexistent. A xylanase, detected by indirect immunofluorescence, was observed at the surface of the vegetative cells, vesicles, or rhizoids. Cross-reactions prove the existence of analogies in structure among the enzymes of these anaerobic gut fungi.
Subject(s)
Fungal Proteins/isolation & purification , Fungi/enzymology , Glycoside Hydrolases/isolation & purification , Rumen/microbiology , Xylosidases/isolation & purification , Animals , Microscopy, Fluorescence , Sheep , Xylan Endo-1,3-beta-XylosidaseABSTRACT
Two beta-endoxylanases produced by Neocallimastix frontalis have been purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. Xylanase I is a nonglycosylated protein with an apparent molecular mass of 45 kDa. Xylanase II is a glycoprotein with an apparent molecular mass of 70 kDa. The pH optima of these enzymes were 5.5 and 6, respectively, and the temperature optimum was 55 degrees C for each enzyme. The endo mode of action of the enzymes was revealed by thin-layer chromatography of xylan hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, confirming the biochemical specificities of the enzymes. Both enzymes exhibited carboxymethyl cellulase activity, and xylanase I was absorbed on crystalline cellulose, indicating that these enzymes might belong to the F family of beta-1,4-glycanases.