Site-specific immobilization of papain on polystyrene beads for the oligo-glutamate synthesis

Yu, L.
Li, C.
Xia, X.
Zhang, F.
Linhardt, Robert J.
Wang, F.
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Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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Site-specific immobilization of papain on polystyrene beads for the oligo-glutamate synthesis, L. Yu, C. Li, X. Xia, F. Zhang, R. J. Linhardt, F. Wang, Applied Catalysis A, General, 630, 118472, 2022.
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 1H NMR showed an average degree of polymerization (DPavg) 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.
Applied Catalysis A, General, 630, 118472
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