Site-specific immobilization of papain on DDI-modified polystyrene beads for the oligo(γ-ethyl-L-glutamate) synthesis

In this paper, a reusable heterogeneous catalyst of papain was prepared and used to catalyze the polymerization for oligo-glutamate. In silico, the 9,9-bis(2,3-epoxypropyl) fluorenyl group (DDI) carried on polystyrene beads (PS-PEG-DDI) showed site-specific binding for sites which are far away the active site of papain. The immobilization of papain on PS-PEG-DDI reached the optimal value of 126 ± 2.5 mg/g beads and 1726.2 ± 34 U/g beads at the papain/beads ratio of 1:5 in phosphate buffer (pH 8.0, 200 mM) at 45 °C for 4 h. The storage stability, pH stability, thermal stability, solvent tolerance and resistance to metal ions of papain were enhanced by the immobilization. The oligomerization of L -glutamic acid diethyl ester hydrochloride via PS-PEG-DDI immobilized papain in phosphate buffer (pH 8, 200 mM) at 45 °C demonstrated the yield of oligo(γ-ethyl-L-glutamate) reached 78.6 ± 1.2% under the conditions of 2 U/ml of papain, 60 mg/ml L -glutamic acid diethyl ester hydrochloride for 4 h. The immobilization increased the yield by about 40%. The analysis by mass spectrometry (MS) and 1 H NMR showed an average degree of polymerization (DP avg ) of product was approximately 7–8, identical to that produced using free papain. The biocatalytic activity of the PS-PEG-DDI immobilized papain remained stable after 5 repeated uses, all giving a yield of over 70%. The reduced activation energy of the reaction catalyzed by the PS-PEG-DDI immobilized papain suggests a more favorable active conformation was probably achieved following immobilization.