Isolation of cellulose and xylan binding proteins
Based on initial cellulose binding studies smaller scale binding experiments were conducted with 25 mg ea of microgranular cellulose and oat spelt xylan. These expts. employed 20 mg/ml of Aspergillus cellulase and Thermomyces xylanase respectively. Polymers were washed with 1.1 ml volumes of 0.2M sodium citrate, pH 5.0. Centrifugations were 1 min at 12K in a microfuge. Oat spelt xylan behaved like microgranular cellulose during binding and washing, but formed a "gel" during phenol extraction. Doubling the volume of phenol failed to resolve the gel, but adding 50 ul of chloroform created a visible organic phase which was withdrawn.
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As the pictures show, cellulose assays were successful and the xylan assay was dubious. In the right hand picture the xylanase conc was higher (35mg/ml), sucrose was added to the xylan phenol extraction (to put the phenol layer on top) and the phenol layer was precipitated with MeOH (since acetone ppts. sucrose). The DNase is obviously of unsatisfactory quality.
Cellulose and chitin binding experiments
Neither sample binds chitin, only Aspergillus binds cellulose
