Covalent immobilization of glucoamylase on 3D-printed poly(lactic acid) carriers for starch hydrolysis in stirred tank reactor

This study presents a novel 3D-printed poly(lactic acid) (PLA) carrier for the covalent immobilization of a commercial glucoamylase. Enzyme carriers were functionalized by aminolysis with ethylenediamine (EDA) and activated with glutaraldehyde. Since covalent immobilization using glutaraldehyde as activating agent involves the formation of imine bonds which are inherently unstable, the use of borohydride as a reducing agent to stabilize these imine bonds after the functionalization step was considered. The highest enzymatic activity (13.68 U g⁻¹ carrier) was obtained when immobilization was performed at pH 10 using NaBH₄ (immobilization yield of 32.80 % ± 0.51 %). Optimal activity conditions were pH 4.92 and 50 °C for the soluble enzyme and pH 5.5 and 50 °C for the immobilized enzyme. The operational stability of immobilized glucoamylase was evaluated for 10 consecutive reaction cycles (of 10 minutes each) and the enzyme maintained 65.19 % of its original activity at the end of the third cycle. Starch saccharification in a stirred tank reactor by immobilized glucoamylase reached 95 % conversion after 12 h and 74 % conversion after 12 h in the first reuse cycle. These findings demonstrate the potential of PLA for glucoamylase immobilization, offering promising prospects for efficient and sustainable starch hydrolysis in industrial enzymatic processes.