We investigated fractionation of cellulase to remove the
β-glucusidase (BG) fraction using an affinity column technique for the purpose of cellobiose production.
First we tried to remove BG from original cellulase using three kinds (KC-flock, Pulp-flock and Avicel) of cellulose powder columns.
When the cellulase was fractionated using the KC-flock as an adsorbent in the column and a 0.1 M acetate buffer (pH 5.0) as an eluent, both endo-
β-1,4-glucanase (EG) activity peak and cellobiohydrolase (CBH) activity peak were overlapped at an initial stage of the fractionation, but the BG activity peak is very low throughout the eluting range. The BG fraction adsorbed on the column could be eluted with a 10 mM sodium hydroxide solution.
When the cellulase was fractionated using the Pulp-flock column and the Avicel column by acetate buffer, BG was not adsorbed on either column. Then, the temperature of the columns was raised to 55°C, and elution by the same buffer was continued. A fraction with CBH activity, EG activity and very low BG activity was obtained by the process.
Secondly, we hydrolyzed the same three kinds of cellulose powders used for columns, using the fractionated enzymes. The soluble reducing sugar contained glucose and cellobiose, and the cellobiose content in the product was over 50 mol% for each of the substrates. The fractionated enzyme that eluted with acetate buffer from the Avicel column at 55°C gave the highest cellobiose contents (over 80% by weight) and this way was the most effective for the production of cellobiose in this study.
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