An indispensable enzyme in saccharification of starch is glucoamylase, (1,4-a-D-Glucan glucohydrolyase EC 3.2.1.3). Many attempts have been made to immobilize this enzyme [1, 2]. Among the immobilization methods physical immobilization offers and advantage of easy reloading of enzyme and it is less harsh to native structure of enzyme. In this report we report the improvement of the performance of Amberlite IRA-904 immobilized glucoamylase by using substrates of low molecular sizes and by reducing particle size of the glucoamylase immobilized resin.
Experimental
2.1 Materials
Glucoamylase (Spiriamylase ยฎ 150L) was from NOVO Industries (Denmark). Amberlite IRA-904, soluble starch and maltose were from BDH Chemical Company (England). Dextrinized starch was prepared from starch [3].
2.2 Immobilization of glucoamylase to Amberlite IRA-904
Amberlite IRA-904 (10 g) was shaken at 150 rpm with 900 mg of glucoamylase protein (9500 U activity) at 30 ยฐC for 1.0 h and filtered through a sintered glass funnel. One Unit (U) of glucoamylase was the amount of enzyme liberating 1.0 mg of glucose in a min.
2.3 Assay of immobilized glucoamylase
Amberlite IRA-904 immobilized glucoamylase (0.5 g) was incubated for 10 min with 15.0 ml of 2.0 % (w/v) starch -0.01 M acetate buffer solution (pH 4.5) at 55 ยฐC and glucose produced was estimated by glucose oxidase method . For soluble enzyme, diluted glucoamylase solution (25 mg protein/7.5 ml 0.01 M acetate buffer, pH 4.5) was shaken with 15.0 ml starch -0.01 M acetate buffer solution (4.0 %, w/v) for 10 min at 55 ยฐC and glucose produced was determined.
2.4 Estimation of protein immobilized to Amberlite IRA-904
Protein immobilized was estimated by three different methods. In indirect method the amount of protein immobilized was considered as the difference between the added enzyme protein and the protein in filtrate obtained after immobilization. The protein was estimated by modified Lowry's method [5]. Secondly protein of glucoamylase immobilized to Amberlite IRA-904 (1.0 g) was also estimated by Kjeldhal method [6]. In the elution method immobilized glucoamylase (1.0 g) was packed in a column and eluted with 3.0 ml of 0.01 M glycine -HCl buffer (pH 2.5). Protein content in the eluate was estimated [5].
2.5 Effect of grinding on the activity of immobilized glucoamylase
Glucoamylase immobilized Amberlite IRA-904 was gently ground in a motor with pestle. Activity of glucoamylase in 0.5 g ground form was determined with (2.0 %, w/v) Starch/Stรคrke 50 (1998) Nr. 2-3, S. 93-95